64 Class Note for MICRB 251 at PSU
64 Class Note for MICRB 251 at PSU
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Notes for BMBMicrb 251 lec 1 21 Molecular amp Cellular Biology I Textbook Molecular Biology of the Cell 4 h Edition Author Alberts et al Instructor Dr B Franklin Pugh Meeting time 12 20 1 10 MWF Meeting place 26 Hosler CHAPTER 1 CELLS AND GENOMES THE UNIVERSAL FEATURES OF CELLS ON EARTH The basic unit of life is the cell Fig 1 1 Most forms of life are just a single cell Humans have 1013 cells all derived from a single cell Cells must consume energy in order to grow and multiply All Cells Store Their Hereditary Information in the Same Linear Chemical Code DNA DNA A T C G each is called a nucleotide computer 0 1 All Cells Replicate Their Hereditary Information by Templated Polymerization Each nucleotide is composed of three parts phosphate sugar and a base Fig 1 2 Only the base is different between A T C G A polymer of nucleotides is called a polynucleotide The unique arrangement of nucleotides forms the genetic code The genetic code provides all the information necessary to make an organism When cells grow and multiply they must duplicate the genetic code The polynucleotide provides a template for its own replication A only pairs with T C only pairs with G All Cells Transcribe Portions of Their Hereditary Information into the Same Intermediary Form RNA In order to use the information in the genetic code the DNA must be read Fig 1 4 Transcription is the process by which parts of the DNA are read Transcription is similar to DNA replication except that an RNA polynucleotide is made DNA A T C G RNA A U C G a U is usedinstead of a T Same pairing rules apply RNA also has an extra 70H group on each sugar RNA and DNA are two different kinds of polynucleotides Different RNAs have different sequences of nucleotides Some RNAs direct chemical reactions more on this later Fig 1 6 Messenger RNA mRNA codes for the production of proteins Translation is the process by which mRNA is read into protein Fig 1 4 All Cells Use Proteins as Catalysts Proteins do almost all the work in the cell Proteins make each cell different Proteins are made up of a linear polymer of amino acids Proteins range in size N1 0071000 amino acids There are 20 different kinds of amino acids The arrangement of the amino acids is dictated by the DNA 9 RNA nucleotide sequence A protein is also called a polypeptide Polypeptides fold up into very precise 3 dimensional structures Fig 1 7a Proteins that catalyze facilitate chemical reactions are called enzymes Fig 1 7b Other proteins also serve as signaling molecules and provide structure to the cell All Cells Translate RNA into Protein in the Same Way Three nucleotides at a time are translated into one amino acid Fig 1 9 A group of three nucleotides that code for an amino acid is called a codon There are 64 possible codons 4X4X4 that code for 20 amino acids Codons are translated by transfer RNAs tRNA The anticodon part of the tRNA pairs with the codon So there are many different kinds of tRNAs Each kind of tRNA is attached to a particular amino acid The ribosome uses mRNA as a template to align the tRNAs which then allows the amino acids to be stitched together Fig 1 10 The ribosome is composed of mostly ribosomal RNA rRNA and ribosomal protein The Fragment of Genetic Information DNA Corresponding to One Protein or One Functional RNA Is One Gene Not all of the DNA has genes Certain stretches of DNA regulate the eXpression of genes When the cell needs to make a particular protein it read or expresses the corresponding ene Thegentire sequence of DNA of an organism is called a genome Life Requires Free Energy Cells take energy food from its environment and use it to build more of itself All Cells Function as Biochemical Factories Dealing with the Same Basic Molecular Building Blocks Part CHAPTER 1 CELLS AND GENOME S All Cells Are Enclosed in a Plasma Membrane Across Which Nutrients and Waste Materials Must Pass The plasma membrane or cell membrane is a sack that keeps all cellular components together It keeps unwanted things out It allows food and other important things to enter Fig 1 13 It is composed of lipids fats A Living Cell Can Exist with Fewer Than 500 Genes Fig 1 14 Humans have over 30000 genes Summary The cell is the minimal operational unit All info to make a cell is stored in DNA Central Dogma DNA makes RNA makes protein Proteins do much of the work in the cell THE DIVERSITY OF GENOMES AND THE TREE OF LIFE Most of life on earth is microorganisms single celled Cells Can Be Powered by a Variety of Free Energy Sources Where do cells get their energy to make more of themselves Inorganic chemicals The sun Other organisms Some Cells Fix Nitrogen and Carbon Dioxide for Others The Greatest Biochemical Diversity Is Seen Among Procaryotic Cells Prokaryotes have no nucleus Fig 1 18 They live in a wide variety of habitats hydrothermal vents Arctic bogs sea dirt other organisms Are microorganisms Eukaryotes have a nucleus Fig 1 43b A nucleus is an intracellular compartment that houses DNA Eukaryotes can be microorganisms or multicellular The Tree of Life Has Three Primary Branches domains Bacteria Archaea and Eucaryotes Fig 1 21 Bacteria and Archaea are prokaryotes Note how diverse the prokaryotes are Note that plants animals and fungi are highly related Some Genes Evolve Rapidly Others Are Highly Conserved When DNA is replicated mistakes are made albeit very rarely Most mistakes mutations have little effect on the organism Some are detrimental to the organism Fig 1 22 Such mutant organisms are eliminated by natural selection abilityinability to thrive or compete with other organisms for survival Therefore the mutation is also lost Such regions of DNA are therefore highly conserved and are indicative of important genetic information In rare cases the mistake changes the genetic code for a protein in a beneficial way This is the core of evolution Most Bacteria and Archaea Have 1000 4000 Genes Natural selection favors those organisms that can reproduce the fastest Small size Small genome Specific environmental niche New Genes Are Generated from Preexisting Genes Fig 1 23 Mutation Nucleotides within a gene can mutate Happens very frequently with HIV as an example Duplication Duplicating a gene allows one to mutate while the other provides the essential function Segment shuffling Two or more genes can be broken up and pieced back together differently Horizontal transfer A gene from one organism can be transferred to a related or unrelated organism Humans have bacterial DNA in their genomesll Gene Duplications Give Rise to Families of Related Genes Within a Single Cell Related organisms have related genes The related genes are said to be orthologs When a gene is duplicated within the same cell allowing them to evolve separately then these genes are paralogs Homologs refer to both orthologs and paralogs All homologs form a gene family Fig 1 26 Genes Can Be Transferred Between Organisms Both in the Laboratory and in Nature Bacterial viruses bacteriophages are mobile genetic vehicles that allow genes to move horizontally Bacteria can also take up DNA from their environment This and the rapid rate of replication allow bacteria to evolve rapidly Fig 1 21 Think antibiotic resistance Horizontal Exchanges of Genetic Information Within a Species Are Brought About by Sex Primordial life may have extensively used horizontal transfer Groups of genes might have moved together Bacteria and Archaea but not Eukaryotes have similar metabolic genes Metabolic genes are involved in getting food Archaea and Eukaryotes but not Bacteria have similar genes that control information ow DNA replication transcription translation Horizontal gene transfer is essentially bacterial sex The Function of a Gene Can Often Be Deduced from Its Sequence Genes with similar sequence have similar function If you know the function of one homolog you then know the function of all homologs More Than 200 Gene Families Are Common to All Three Primary Branches of the Tree of Life Table 1 2 Mutations Reveal the Functions of Genes How do we figure out what the function of any given gene is We know the function of only a small percentage of genes Determine function through biochemistry Isolate the protein coded for by a gene and determine what chemical reaction it carries out Determine function through genetics Mutate the gene and see what effects phenotype it has on the organism e g growth rate Molecular Biologists Have Focused a Spotlight on Model Organisms Bacteria E coli Eukaryote Yeast Arabidopsis plant fruit ies mice and more Summary All life requires energy via inorganic chemicals sunlight or other organism Prokaryotes represent the bulk of life s diversity and mass on earth There are three domains of life bacteria archaea and eukaryotes All life evolved through mutation duplication shu ling and horizontal transfer of genes GENETIC INFORMATION IN EUCARYOTES Humans are eukaryotes so we have an interest in how eukaryotes work Eukaryotes are much more complex than prokaryotes But not more evolved Eukaryotes have more complex genomes cell organization and can be multi cellular Eucaryouc Cells May Have Or1g1nated as Predators A eukaryotic cell is 1000x larger than a prokaryotic cell Fig 1 31 Eukaryotes have a nucleus nuclear membrane or nuclear envelope that compartmentalizes the DNA Eukaryotes also have other internal membrane compartments Eukaryotes have a protein cytoskeleton that gives shape to the cell Fig 1 32 Prokaryotes use a cell wall By rearranging the cytoskeleton eukaryotic cells can rapidly change shape Eukaryotic cells can engulf bacteria think immune system A primitive eukaryoticilike cell might have eaten other bacteria Eucaryotic Cells Evolved from a Symbiosis Eukaryotic cells have mitochondria Fig 1 34 Mitochondria are membrane compartments organelles that convert food energy into usable chemical energy respiration Mitochondria also have a small genome Some eukaryotic cells eg plants have chloroplasts chloroplasts are organelles and have a genome chloroplasts convert light energy into usable chemical energy photosynthesis Mitochondria and chloroplast were once free living bacteria that were engulfed by primitive eukaryotes and formed a symbiotic relationship Fig 1 35 Is mitochondrial and chloroplast DNA more like bacteria or eukaryotes Eucaryotes Have Hybrid Genomes Eucaryotic Genomes Are Big Fig 138 1000x longer than bacteria but only about 20K more genes 99 of the eukaryotic genome does not code for genes Probably just junk When was the last time you removed unnecessary files from your 80 gig hard drive The Genome Defines the Program of Multicellular Development Multi cellular organisms have a diversity of cell types all derived from a single fertilized egg and all having the same genome Humans have skin cells liver cells and brain cells sometimes Plants have leave cells ower cells and root cells Different cell types are made when different subsets of genes are expressed When you are listening to music it s like having a different favorite play lists depending on your mood or what your doing e g breaking up w your boygirlfriend vs having a party 7 maybe Cells are constantly sending signals to each other Cells at different locations get different signals Signals trigger the expression of particular sets of genes favorite play list Fig 1 40 No signal 7 no expression Fig 141 Many Eucaryotes Live as Solitary Cells the Protists A Yeast Serves as a Minimal Model Eucaryote Saccharomyces cereVisiae bakers yeast and brewers yeast Fig 1 43 Fungi Advantages Small genome Easy genetics and biochemistry Rapidly grows and divides Inexpensive Most cellular functions highly conserved with humans The Expression Levels of All The Genes of An Organism Can Be Monitored Simultaneously As scientists we used to study one gene at a time Now that the entire yeast genome has been sequenced we can study the expression of all 6300 yeast genes at a time using DNA microarrays Fig 1 45 Each spot corresponds to the DNA of a particular gene 6300 spots for yeast mRNA from a particular gene will bind hybridize to it s cognate spot If we color the mRNA first the spot turns color To see how the yeast genetic program changes when they are hungry vs when they are well fed glucose Isolate mRNA from hungry cells and color it red Isolate mRNA from fed cells and color it green Mix both together and hybridize to the DNA spots Red spots mean the gene was turned on in hungry cells and off in fed cells What do the green yellow and black spots mean 10 The World of Animal Cells Is Represented By a Worm a Fly a Mouse and a Human worm Caenorhabditis elegans fly Drosophila melanogaster mouse Mus musculus human Homo sapien Studies in Drosophila Provide a Key to Vertebrate Development The Vertebrate Genome Is a Product of Repeated Duplication Genetic Redundancy Is a Problem for Geneticists But It Creates Opportunities for Evolving Organisms Mutating a gene might knock out its function but if another gene serves the same function there will be no phenotype Gene duplication provides great opportunities for the duplicated gene to evolve new functions Fig 1 51 The Mouse Serves as a Model for Mammals Able to knock out specific genes We Are All Different in Detail Mouse Human 90 identical Chimp Human 99 Human Human 999 Summary Eukaryotes are evolutionary less diverse than prokaryotes but are way more complex Eukaryotic cells arose by symbiosis with prokaryotes Playing different parts of the genome gives rise to different cell types Model organisms are used as experimental surrogates to humans The more complex the organism the harder it is to work with but may be a better proxy for humans Part II Introtltmct om Um em 11 CHAPTER 2 CELL CHERHarm ND BIOSYNTHESIS CHAPTER 2 CELL CHEMISTRY AND BIOSYNTHESIS THE CHEMICAL COMPONENTS OF A CELL Cells Are Made of Relatively Few Types of Atoms gt99 of living matter is composed of six elements P S C O H N COHN represent Ng7 Other important elements Cl Ca Mg Si Zn Co Mn Fe Se and others The Outermost Electrons Determine How Atoms Interact Ionic Bonds Form by the Gain and Loss of Electrons Know how a covalent bond differs from an ionic bond Fig 2 5 Covalent bonds are very strong and stable essentially riveting atoms together Ionic bonds a positive charge interacting with a negative charge Ionic bonds are weak in water because water interacts with the positive and negative charges making it hard for them to interact with each other Fig 214 Ions have gained or lost a charge electron Cations are positively charged ions Anions are negatively charged ions Covalent Bonds Form by the Sharing of Electrons Covalent bonds are strong Atoms form molecules through covalent bonds Covalent bonds also link repeating units of a polymer together Defined as a stable chemical link between two atoms produced by sharing one or more pairs of electrons The amount of energy required to break a covalent bond varies depending on atoms and environment ave 90 kcalmol Enzymes are required to break covalent bonds under normal physiological conditions There Are Different Types of Covalent Bonds Polar vs nonpolar An Atom Often Behaves as if It Has a Fixed Radius Different representation of molecules Fig 2 12 12 Water Is the Most Abundant Substance in Cells 70 of a cell is water Some Polar Molecules Form Acids and Bases in Water Four Types of Non Covalent Interactions Help Bring Molecules Together 1n Cells Table 2 2 and Panels 2 2 and 2 3 on pp 112 1 15 Ionic bonds Weak relative strength 3 Cohesion between a positively charged atom and a negatively charged atom Fig 214 Water and salts are polar or charged and so can compete with these interactions thereby weakening them By measuring the interactions between two molecules as a function of salt NaCl KCl etc conc one can get a quantitative handle on the extent of ionic interactions Examples DNA phosphates and protein lysine side chains make ionic bonds Protein side chains glutamate and arginine make ionic interactions Hydrogen bonds Weak relative strength 1 Hydrogen atom with partial positive charge interacts with two electronegative atoms Fig 215 Two electronegative atoms such as N and O can share a hydrogen atom even though a H atom can only form a single covalent bond Water can compete with Hibonds thereby weakening them Example Protein secondary structure DNA baseipairing van der Waals interactions Very weak relative strength 01 A large number of them can add up to generate strong interactions Due to asymmetric electrical charges two atoms at very close distances will attract each other Handiiniaiglove fit Hydrophobic interactions Water interacts with itself via hydrogen bonds surface tension Nonpolar groups cannot interact with water and so are excluded oil and water don39t mix Water exclusion causes nonpolar groups to self associate Example Interior of proteins Two surfaces of proteins 13 ID BIOSYNTHESIS 11 Enimtlueft orm th CHAPTER 2 CELL CHEMISTRX A Cell Is Formed from Carbon Compounds Carbon represents the core constituent of all life because it can make a variety of strong covalent bonds with other elements Chemical groups Functional groups Panel 2 1 on page 111 CH3 methyl OH hydroxyl COO carboxylate CO carbonyl NH2 amino amine SH2 sulfhydryl PO3 phosphate Cells Contain Four Major Families of Small Organic Molecules Fig 2 17 Sugars Fatty acids Amino acids Nucleic acids Sugars Are Energy Sources for Cells and Subunits of Polysaccharides Fig 2 18 1920 Panel 2 4 pp 116 1 17 o Monosaccharides gt disaccharides gt oligosaccarides gt polysaccharides o Hexose pentose triose 0 Be able to distinguish glucose from ribose from glycerol 0 Structure linear branched ring 0 Functional groups OH C20 0 Sugars have a variety of functions food 7 glucose sucrose glycogen sancn a carbon sugars or hexoses nucleic acid backbone e ribose 5 carbon ce11 adhesion ce11 recognition 39 cell wall Part ll ll ti CHAPTER 2 CELL CilEiJiiS fi 14 ID Fatty Acids Are Components of Cell Membranes Fig 22122 Panel 2 5 pp 118 1 19 o Amphiphillic hydrophobic repeat of CH2 0 One end terminates with a carboxylate eg palmitate o Phospholipids end with phosphate 0 Be able to recognize fatty acids phospholipids triglycerides glycolipids 0 Fatty acids have a variety of functions food 7 stored as triglycerides fat droplets in cytoplasm cell membmue and mm membmues consists ofphospholipids 2 FA phosphate head group lipid bilayer imam signaling Amino Acids Are the Subunits of Proteins Fig 223 24 0 Functional groups carboxylate central carbon alpha amino 0 Be able to distinguish amino acids gt peptides gt polypeptides proteins 0 Know what a peptide bond amide linkage is 0 There are 20 natural amino acids 0 They differ by the functional groups or side chain arrayed off of the alpha carbon 0 Know the properties of the side chains nonpolar hydrophobic acidic basic polar uncharged 0 Amino acids chart 0 Amino acids serve a variety of functions enzymatic catalysis cell structure 39 energy source cellrcell signaling 39 toxins 15 OSYNTV AND Bl Nucleotides Are the Subunits of DNA and RNA Fig 226 27 28 Panel 2 6 pp120 121 O O 0 Composition phosphate ribose sugar pyrimidine purine base DNA 2 H vs RNA 2 OH Purine vs pyrimidine Four bases adenosine A guanosine G cytosine C thymine T DNA or uracil U RNA N glycosidic bond Nucleoside base sugar vs nucleotide base sugar phosphate phosphodiester linkage nucleotide gt oligonucleotide gt polynucleotide base pairing A w TU G w C Nucleotides serve a variety of functions genetic information stong DNA RNA carriers of chemical energy high energy phosphoauhydride bonds ATP 39 enzyme cofactm39s CoA NAD signaling molecules cyclic AMP The Chemistry of Cells is Dominated by Macromolecules with Remarkable Properties Noncovalent Bonds Specify Both the Precise Shape of a Macromolecule and its Binding to Other Molecules Fig 232 Summary Cells are composed of primarily six elements PSCOHN Covalent bonds stably connect atoms to form molecules to form biopolymers Ionic hydrogenbonds van der Waals and hydrophobic interactions drive dynamic interactions between and among biopolymers and small molecules Specific interactions are provided through precisely positioned functional groups on the 3D structure of the biomolecules Sugars are the building blocks of polysaccharides Fatty acids are the building blocks of membranes Amino acids are the building blocks of proteins Nucleotides are the building blocks of DNA and RNA Part ll introtlmt on to the 16 CHAPTER 2 CELL CHEMISTRY AND BIOSYNTHESIS The rest of this chapter will be covered in more detail in the second half of BMB 251 F or now just read the text relevant to the figures CATALYSIS AND THE USE OF ENERGY BY CELLS Cell Metabolism Is Organized by Enzymes Fig 2 34 Every enzyme has one particular function division of labor A cell can have many copies of the same enzyme Biological Order Is Made Possible by the Release of Heat Energy from Cells Enzymes Lower the Barriers That Block Chemical Reactions Fig 2 44 46 Fig 2 46c doesn t make any sense Breaking covalent bonds requires activation energy Enzymes lower the activation energy Enzymes bind to substrates and convert them to products How Enzymes Find Their Substrates The Importance of Rapid Diffusion Life processes require molecules to interact They do so via random diffusion or active transport Diffusion is temperature dependent Fig 2 48 The distance coverage only goes up by the square root of the allowed time Large molecules diffuse slower than small molecules Long distances movements may involve active transport Active transport requires the input of energy Diffusion limited reactions occur as fast as the molecules collide More concentrated the fast the reaction goes Why are many reactions not diffusion limited Nonproductive collisions Conformational changes in the protein Catalytic steps Dissociation of a regulatory subunit or molecule The cell is very crowed with biopolymers and other biomolecules Fig 2 49 17 AND BIOSYNT Activated Carrier Molecules are Essential for Biosynthesis When cells take in food they break down the bonds which releases energy Some of the energy dissipates as heat Some of the energy is coupled to the production of activated carriers Activated carriers have high energy covalent bonds that can be used to make an unfavorable reaction more favorable Important energy carriers ATP NADH and NADPH The Formation of an Activated Carrier Is Coupled to an Energetically Favorable Reaction Fig 2 56 High energy covalent bonds are very unstable and thus their easy breakage can be coupled to the breakage of more stable covalent bonds ATP Is the Most Widely Used Activated Carrier Molecule Fig 2 57 Be able to recognize ATP and distinguish it from ADP Energy Stored in ATP Is Often Harnessed to Join Tvvo Molecules Together Fig 2 59 ATP adenosine Lriphosphate is hydrolyzed to ADP denosine diphosphate Hydrolysis as its name implies means using water to break bonds Know the ATP hydrolysis reaction There Are Many Other Activated Carrier Molecules in Cells The Synthesis of Biological Polymers Requires an Energy Input Example of polynucleotide synthesis Fig 2 67 Summary Cellular reactions eg synthesis of more cell components are carried out by enzymes Energetically unfavorable reactions can be coupled to favorable ones ATP is the major energy carrier in the cell equivalent to in our economy ll T frotluct on f 18 CHAPTER 2 CELL CHEMISTRY A ND BIOSYNTHESIS HOW CELLS OBTAIN ENERGY FROM FOOD Food Molecules Are Broken Down in Three Stages to Produce ATP Fig 2 70 1 Enzymatic break down of food Covalent bonds are broken such that biopolymers are broken down to monomer units eg glucose This happens in the intestines and is relevant to multiicellular organisms Monomer units are absorbed into the cell Transport proteins help the molecules traverse the membrane The molecules end up in the cell s cytoplasm 2 Glucose is cleaved into two molecules called pyruvate This occurs over many steps Withmany chemical intermediates A series of enzymes catalyze these coupled reactions ATP input is used to help lower the activation energy Ultimately more ATP produced The Whole cascade of events is called glycolysis Pyruvate diffuses into mitochondria Where it becomes a substrate for respiratory enzymes 3 Pyruvate is converted to an activated molecule called acetyl coA A series of very complex enzymatic reactions couples the break down of acetylicoA to the production of ATP Summary Food is broken down into monomer units Breakage of covalent bonds release energy which is recouped in the form ofATP and other energy carriers 19 NS CHAPTER 3 PROTEINS THE SHAPE AND STRUCTURE OF PROTEINS The Shape of a Protein Is Specified by Its Amino Acid Sequence A protein is made from a polymerized chain of amino acids The covalent bond that links amino acids is called a peptide bond Fig 3 1 Small chains of amino acids lt100 are called peptides Long chains are called polypeptides Polypeptides and proteins mean the same thing but often polypeptide refers to the unfolded chain There are 20 different amino acids Fig 3 2 The part that is similar among all 20 is calledthe backbone The part that is unique is called the side chain Be able to recognize amino acid names Know that some have basic acidic or nonpolar side chains Each type of amino acid serves a purpose in the context of a protein Amino acids with similar side chain properties have similar function If you are a serious BMB major then you should memorize all 20 amino acid names and properties and be able to identify their side chains It ll be needed to really understand 251 and related courses Fig 373 and Panel 371 A polypeptide chain folds back on itself giving the protein a unique 3 D structure Four noncovalent forces direct the folding Fig 3 5 3 6 3 7 Ionic hydrogenibonding van der Waals and hydrophobic Proteins Fold into a Conformation of Lowest Energy Fig 3 8 Although the sequence of amino acids dictate the folding pathway and the final folded conformation in the cell other proteins called chaperones assist the folding process Proteins range in size from 50 2000 amino acids Large proteins have multiple independent folding domains Fig 3 12 Domains can be thought of as different proteins strung together more below The on Helix and the 5 Sheet Are Common Folding Patterns Fig 3 9 The Protein Domain Is a Fundamental Unit of Organization There are four levels of organization Primary structure is the linear arrangement of amino acids Secondary structure involve a helices and b sheets The protein can be partially unfolded and still have secondary structure Tertiary structure represents the full 3 D structure of a protein Fig 3 12 3 13 A protein can have one or more independently folding domains Domains range from 5350 amino acids Quaternary structure refers to complexes of multiple proteins So quaternary interactions involve more than one protein Tertiary structure of a multi domain protein is analogous to quaternary structure 20 Few of the Many Possible Polypeptide Chains Will Be Useful Certain amino acids along the polypeptide chain are more important than others in determining the structure of a protein Proteins Can Be Classified into Many Families Proteins of similar biochemical function are likely to have similar structure Fig 3 14 However the proteins may or may not have nearly identical primary sequence Fig 3 15 Proteins Can Adopt a Limited Number of Different Protein Folds Sequence Homology Searches Can Identify Close Relatives Fig 3 17 Computers can be used to align the primary sequence of proteins to determine if they are related Computational Methods Allow Amino Acid Sequences to Be Threaded into Known Protein Folds Some Protein Domains Called Modules Form Parts of Many Different Proteins Some proteins may be unrelated except in one domain or module Fig 3 18 3 19 3 21 Remember genes can evolve by shuf ing parts of the gene Since we might not know if a conserved region as defined by comparing primary sequence meets the definition of a domain independently folding unit we call them modules Some proteins may be related only by a motif A motif is a small sequence of amino acids found in many proteins A motif is small than a domain and probably does not fold independently of the rest of the protein Motifs typically represent an interface 7 a section of the protein that binds something else like another protein or a small molecule The Human Genome Encodes a Complex Set of Proteins Revealing Much That Remains Unknown Larger Protein Molecules Often Contain More Than One Polypeptide Chain Proteins are generally thought of as having one polypeptide However many proteins have more than one polypeptide and can be thought of as protein complexes Fig 3 21 3 22 3 23 3 24 Each polypeptide of a protein complex is called a subunit Some Proteins Form Long Helical Filaments Fig 3 25 3 26 21 A Protein Molecule Can Have an Elongated Fibrous Shape Extracellular Proteins Are Often Stabilized by Covalent Cross Linkages Fig 3 28 3 29 Disulfide bonds between cysteine side chains stabilize protein tertiary and quaternary structure Protein Molecules Often Serve as Subunits for the Assembly of Large Structures Advantages of using repeating identical subunits to build very large protein structures Less genetic information required Assembly and Disassembly can be easily controlled since interactions are repeated Single domain small subunits allow mis folded subunits not to be incorporated Some very large protein structures with repeated subunits Fig 3 30 Actin filaments Tubulin filaments Bacterial agella Some super sized structures Fig 3 31 3 32 Viral coats capsids Many Structures in Cells Are Capable of Self Assembly The Formation of Complex Biological Structures Is Often Aided by Assembly Factors Summary The sequence of amino acids dictates the structure of a protein Proteins have substructure including a helices and 3 sheets a helices and 3 sheets fold into domains Domains fits together to form the protein Certain proteins can coalesce to form complexes or large structures Proteins with similar function will have similar structure and may or may not have similar amino acid sequence i ll Entrod quot 22 CHAPTER3 PROl r I PROTEIN FUNCTION All Proteins Bind to Other Molecules Fig 3 37 Molecules that bind to proteins are called ligands Proteins are very selective toward the ligands they bind Precise docking of the ligand provides specificity Driving forces ionic hydrogen bonding van der Waals hydrophobic Fig 3 38 3 43 The Details of a Protein s Conformation Determine Its Chemistry Amino acid side chains can be made to be very reactive Good for catalyzing biochemical reactions Sequence Comparisons Between Protein Family Members Highlight Crucial Ligand Binding Sites Fig 3 40 Binding Strength Is Measured by the Equilibrium Constant Fig 3 44 Enzymes Are Powerful and Highly Specific Catalysts Enzymes do not get used up in the reaction Enzymes do not alter the equilibrium ratio of substrate and product Enzymes speed up reaction rates Enzymes can catalyze the reverse reaction as well ltl o1 o 23 39 tOT E N Substrate Binding Is the First Step in Enzyme Catalysis The reaction that an enzyme catalyzes occurs in the enzyme active site 0 The 3 dimensional arrangement of amino acids in the active site defines the active site The substrate andor ligand precisely dock at the active site via noncovalent and sometimes covalent interactions Some ligands bind at other sites on the protein and change the proteins conformation and activity 39 These other sites are called allosteric sites The interplay of amino acid side chains in the active site can cause a side chain to be hyper reactive The first step in an enzyme catalyzed reaction is the binding of a substrate to the enzyme s active site 0 O O In this example a bond it being broken e g ATP gt ADP phosphate E S ltgt ES ltgt EP ltgt E P E enzyme S substrate P 2 products The substrate encounters the active site via random diffusion Most of the time the substrate passes right by the active site or hits the protein at the wrong site Fortunately diffusion is rapid and so a molecule might make over a billion collisions with the enzyme every second Of course in the cell there are many different kinds of molecules which also collide with the enzyme The wrong molecule might enter the active site Precise docking of the substrate in the active site keeps it there But sometimes the substrate dissociates before it can go onto the second step How often a substrate dissociates versus going on to step 2 depends upon how strong the interaction is between the protein and the substrate When a substrate binds an active site it often induces the protein into a subtle change in conformation 24 The second step is catalysis E S ltgt ES ltgt EP ltgt E P O O The reason why the substrate does not spontaneously convert to product in the absence of the enzyme is there is a major energy barrier in breaking covalent bonds This is called the activation energy When the substrate is halfway to product its bond is very strained This can cause a slight change in the conformation of the substrate This state is very unstable and it called the transition state The enzyme active site is actually configured to bind the transition state much better than to the substrate So the substrate is chemically stable but binds to the active site relatively weakly The transition state which is chemically very unstable binds to the active site very strongly This results in the enzyme lowering the activation energy for the reaction by stabilizing the transition state As a dramatic proof of principle on this antibodies raised against synthetic artificially stable transition state analogs can be used to catalyze the native reaction The third step in the reaction is the dissociation of the products 0 O E S ltgt ES ltgt EP ltgt E P In order for the enzyme to catalyze another reaction the product must first dissociate from the active site Since in many cases the product is not that much different from the substrate it is important that neither the substrate nor the product have too high of an affinity for the enzyme Otherwise it would get stuck A complete reaction cycle is called a turnover The turnover number is the number of reactions an enzyme can catalyze per unit time eg 300 per sec 25 How fast an enzyme works depends on the concentration of substrate 0 O O O O O 0 At low S but S gtgt E V reaction velocity is proportional to S V has units of molar per sec M s l As S increases it more frequently encounters an enzyme that already has an S bound The enzyme becomes saturated with S As E gt saturation V gt Vmax called vee max Turnover number Vmax E KM S at which reaction proceeds to half Vmax KM is a measure of substrate affinity as well as its tendency to react O O Substrates in the cell are often at concentrations in the Km range Slight changes in the enzymes Km caused by allosteric ligands can cause dramatic changes in the amount of product produced Enzymes Speed Reactions by Selectively Stabilizing Transition States Tightly Bound Small Molecules Add Extra Functions to Proteins Where amino acid side chains are insufficient coenzymes are employed 0 O O O O Coenzymes are not proteins Coenzymes are often but not always derivatives of nucleotides Remnants of an ancient RNA world Many vitamins are precursors to coenzymes Biotin thiamine Coenzymes are very high affinity ligands Other small molecules add functionality O O Metals zinc iron Many more Multi enzyme Complexes Help to Increase the Rate of Cell Metabolism Fig 3 54 Product of one reaction becomes the substrate for the next Think of a handioff in a relay race No need for diffusion 26 The Catalytic Activities of Enzymes Are Regulated Fig 3 55 3 56 Feed back inhibition Product of one reaction inhibits the production of the substrate for that reaction When you eat and get full and don t feel like eating anymore this is like feed back inhibition Allosteric Enzymes Have Two or More Binding Sites That Interact Fig 3 57 3 58 An allosteric site binds a ligand changing the conformation of the active site an protein Tvvo Ligands Whose Binding Sites Are Coupled Must Reciprocally Affect Each Other s Binding Symmetric Protein Assemblies Produce Cooperative Allosteric Transitions Fig 3 60 See movie 39 on Cell Biology Interactive to get a better feel for the dynamics of allosteric regulation Many Changes in Proteins Are Driven by Phosphorylation Protein kinases add phosphates to proteins called phosphorylation Fig 6 63 Phosphate is derived from ATP Amino acid side chains that can get phosphorylated have OH groups Tyrosine Serine Threonine Protein phosphatases remove phosphates from proteins Done via hydrolysis A Eucaryotic Cell Contains a Large Collection of Protein Kinases and Protein Phosphatases Cells contains hundreds of different kinds of kinases and phosphatases Each is very specific for a set of proteins Phosphorylationdephosphorylation serves as a molecular switch turning on and off the activity of a protein Phosphorylationdephosphorylation events are dynamic The Regulation of Cdk and Src Protein Kinases Shows How a Protein Can Function as a Microchip Fig 3 66 See movie 158 on Cell Biology Interactive to get a better feel for this Get a sense of the major regulatory themes and not specific names 27 Proteins That Bind and Hydrolyze GTP Are Ubiquitous Cellular Regulators Fig 3 70 Some proteins are activated when they bind GTP eg Ras The GDP bound protein is inactive Regulatory Proteins Control the Activity of GTP binding Proteins by Determining Whether GTP or GDP Is Bound Regulatory proteins eg GAP can induce GTP hydrolysis in the GDP bound protein Fi 371 Gfanine nucleotide exchange proteins induce GDP to dissociate thereby allowing the protein eg Ras to be reactivated See movie 159 on Cell Biology Interactive to get a better feel for this Get a sense of the major regulatory themes and not specific names You must know the major intracellular signaling pathways Fig 3 72 Motor Proteins Produce Large Movements in Cells Fig 3 75 3 76 ATP hydrolysis converts a random walk into a directional walk ATP hydrolysis is coupled to conformational changes in the protein that allow forward but not backward movement Any backward movement must be coupled to ATP synthesis which is energetically unfavorable but could be accomplished if the ADP and phosphate concentrations were high enough and the ATP concentration low enough Examples of directional movement Muscle contraction Mitosis Cell migration Pumping ions eg sodium out of a cell DNA polymerization Protein synthesis Membrane bound Transporters Harness Energy to Pump Molecules Through Membranes Also the reverse is true Movement of ions specifically hydrogen ions through a membrane is coupled to ATP synthesis See movie 141 on Cell Biology Interactive to see an awesome example See also 142 if you want to know what grad students do with their spare time Proteins Often Form Large Complexes That Function as Protein Machines We be seeing lots of this over the next several weeks DNA replication Genetic recombination Transcription Translation RNA splicing Part ll introduction to 28 CHAPTER 3 PROTEINS A Complex Network of Protein Interactions Underlies Cell Function Summary Proteins are designed to bind other molecules to elicit some biological change Enzymes are a type of protein that catalyze biochemical reactions To catalyze biochemical reactions enzymes bind substrates make or break covalent bonds and release products Proteins are regulated by the binding of small molecules other proteins or by phosphorylation These molecules cause their protein targets to change shape making them more or less active Enzymes can use ATP hydrolysis to get work done llT a quotJeri a 29 CHAPTER 4 DNA AND CHROlleSlel Part II Basic Genetic Mechanisms CHAPTER 4 DNA AND CHROMOSOMES THE STRUCTURE AND FUNCTION OF DNA A DNA Molecule Consists of Two Complementary Chains of Nucleotides A DNA double helix consists of two antiparallel polynucleotide chains Fig 4 3 The two chains are held together by hydrogen bonding of bases A base pairs with T C base pairs with G A sugar phosphate base is called a what Must know how to identify antiparallel strands Must know what 5 to 3 direction is o The sugar consist of a four carbon called 139 239 339 and 439 and one oxygen pentameric ring plus an extra carbon 5 and numerous OH hydroxyl groups hanging off the ring 0 Each sugar is linked by a phosphodiester bond attached at the 3 and 5 position 0 The chain therefore has directionality 0 You are moving in the 5 to 3 direction when you go from the 5 carbon to the 439 carbon to the 339 carbon on the same sugar 0 What comes after the 339 carbon The Structure of DNA Provides a Mechanism for Heredity In Eucaryotes DNA is Enclosed in a Cell Nucleus Summary Genetic information that defines an organism is carried in a linear sequence of nucleotides DNA is double helix of two antiparallel strands Apairs with T and C with G Part M CHAPTER 4 rm 30 CHROMOSOMAL DNA AND ITS PACKAGING IN THE CHROMATIN FIBER Eucaryot1c DNA Is Packaged mto a Set of Chromosomes F1 g 4 10 One chromosome corresponds to one continuous DNA double helix Humans have 24 different chromosomes Two sets of 22 different autosomal chromosomes Males have one each of seX chromosomes X and Y Females have two X chromosomes So humans have a total of 46 chromosomes in every cell A chromosome can be as long as a hundred million nucleotides Eukaryotic chromosomes are linear Bacterial chromosomes are circular one end is connected to the other end Chromosomes Contain Long Strings of Genes The Nucleotide Sequence of the Human Genome Shows How Genes Are Arranged in Humans Fig 4 15 The human genome has been sequenced A composite of seven ethnically diverse people It contains over 3 billion base pairs and over 30000 genes There could be as many as 60000 genes but we don t yet know how to recognize them Genes are arranged linearly along chromosomes Genes are split into pieces consisting of exons The DNA between exons is called introns Exons are about 5 of the length of introns Much of human DNA contains repetitive elements Fig 4 17 Less than 2 of the human genome codes for protein Comparisons Between the DNAs of Related Organisms Distinguish Conserved and Nonconserved Regions of DNA Sequence Since exons comprise lt2 of the genome and since we do not know how the DNA sequence dictates where exons start and stop we don t really know what is coding and what is noncoding Introns are often considered to be junk since they are of little importance and are generally not conserved By comparing aligning the human genome with the mouse genome we should be able to determine what regions are conserved Conserved regions are likely to be important Humanmouse other comparisons reveal that the linear arrangement of genes along a chromosome is not static Fig 4 19 Large chunks move from chromosome to chromosome 7 31 CHAPTER 4 DNA AN D CHROlleSChlEB Chromosomes Exist in Different States Throughout the Life of a Cell Cells grow and divide This repeated process is called the cell cycle more on this later Fi 4 20 At ne stage of the cell cycle Sphase the chromosomes are replicated At the next stage mitosis the chromosomes get compacted and packaged so that they can be delivered to both cells after cell division These condensed chromosomes are what is normally shown in textbooks Normally chromosomes are decondensed and therefore barely visible by even the most powerful microscope Fig 4 21 Genes are expressed when the chromosomes are decondensed Each DNA Molecule That Forms a Linear Chromosome Must Contain a Centromere Two Telomeres and Replication Origins Replication origin locations along the chromosome where the replication machinery initiates chromosome duplication Centromere a region of DNA the mitotic spindle attaches so that it can drag the chromosome to the daughter cells during cell division Telomere DNA sequences that act as caps protecting the ends of chromosomes Fig 4 22 Sometimes chromosomes can get damaged like when they break The cell has machinery to repair broken chromosomes Remember that normal chromosomes are linear The ends of normal chromosomes would be recognized as broken if not for telomeres DNA Molecules Are Highly Condensed in Chromosomes Parts of chromosomes are packaged to varying degrees The level of packaging is quite dynamic re ecting the need to accesssequester the genetic information Nucleosomes Are the Basic Unit of Eucaryotic Chromosome Structure Chromosomes are normally covered with many different proteins each having a different role in managing the genetic information The DNA plus these proteins is generally referred to as chromatin Fig 4 23 The major proteins that packages DNA are called histones Histone H2A histone H2B histone HB and histone H4 These are collectively referred to as core histories Several histones get together to form a protein complex in which 150 200 base pairs of DNA wrap around Fig 4 25 The histone complex plus the DNA is called a nucleosome Actually 146 bp base pairs are in contact with the histories N50 bp form a linker between adjacent nucleosomes The beginning part of each histone polypeptide chain reach out like arms and help regulate the accessibility of the DNA Called aminoterminal tails Fig 4 32 Nucleosomes make chromosomal DNA look like beads on a string The Structure of the Nucleosome Core Particle Reveals How DNA Is rtll z CHAPTER 4 DNA AND CHROlVlOSChlEB 32 Packaged Fig 425 Nucleosomes Are Usually Packed Together into a Compact Chromatin Fiber Fig 4 30 The beads on a string 10 nm ber are usually compacted further by histone H1 to form a 30 nm ber The chromatin fiber toggles between the 10 and 30 nm fiber when it is generally decondensed not in mitosis and the genetic info needs to be read or not read Fig 4 31 The amino terminal tails of the core histones may also contribute to formation of the 30 nm fiber Fig 4 32 ATP driven Chromatin Remodeling Machines Change Nucleosome Structure To access the genetic information the histones must be moved or removed Chromatin remodeling complexes move and or remove histones Fig 4 33 Chromatin remodeling complexes control gene expression Fig 4 34 There are different kinds of chromatin remodeling complexes Some use the energy of ATP hydrolysis to move histones around Covalent Modification of the Histone Tails Can Profoundly Affect Chromatin Fig 4 35 The core histone amino terminal tails are subjected to covalent modification Covalent modification alters what the tails can do to the DNA resulting in a different functional state of the chromatin ie accessible vs not accessible Note that DNA is negatively charged phosphate backbone Histones are positively charged lysine and arginine side chains Modified histone tails lose their positive charge What is the consequence of this with regard to histone 7 DNA interactions Lysine side chains are acetylated by histone acetyltransferases called HATS HAT are enzymes that convert lysine to acetyl lysine Enzymes that remove the acetyl group are called histone deacetylases HDACs Other enzymes can methylate the lysines Others phosphorylate serines on the tail Proteins bind to modified histone tails Summary Eukaryotic DNA resides in a group of polynucleotide chains called chromosomes Genes are scattered and fragmented through the chromosomes covering lt2 Chromosomal DNA is packaged into chromatin by histones forming nucleosomes Chromosomes are continuously condensing compacting and decondensing for purpose of packaging and transport mitosis and for accessingsequestering genetic information Accessibility is regulated by enzymes that modify chromatin by physically moving histones or Part 11 Basic Genetic Mechanisms CHAPTER 4 DNA AND CHROMOSOMES by covalently modifying histones 33 M Erratic 34 CHAPTER 4 DNA AND CHROMOSOMES THE GLOBAL STRUCTURE OF CHROMOSOMES Heterochromatin Is Highly Organized and Usually Resistant to Gene Expression Highly condensed chromatin that is generally devoid of genes However heterochromatin does play important roles in chromosome maintenance Genes that are placed in heterochromatin by scientist are generally in active The same gene place in normally active euchromatin is active Mitotic Chromosomes Are Formed from Chromatin in Its Most Condensed State Fig 4 55 Individual Chromosomes Occupy Discrete Territories in an Interphase Nucleus Fig 4 60 Summary Chromatin structure is quite diverse and largely unknown Part III 35 CHAPTER 5 DNA REPLICATION REPAIR AND REC OMBINATION CHAPTER 5 DNA REPLICATION REPAIR AND RECOMBINATION THE MAINTENANCE OF DNA SEQUENCES DNA encodes all the information necessary to make an organism In order for an organism or cell to reproduce it must make a nearly exact copy of its DNA I said nearly since mistakes during DNA replication are the essence of evolutionary change A permanent change in the DNA is called a mutation Mutation Rates Are Extremely Low E coli makes a permanent mistake mutation about once every billion nucleotides of replicated DNA Many Mutations in Proteins Are Deleterious and Are Eliminated by Natural Selection Low Mutation Rates Are Necessary for Life as We Know It Mutations at very low rates are essential for evolution However high mutation rates in germ cells are detrimental to the species Germ cells are sperm and egg which go to make the neXt generation High mutation rates in somatic cells cause a variety of diseases including cancer Somatic cells are all nonigerm cells like skin cells liver cells brain cells etc Cancer is an uncontrolled proliferation of cells Your body has builtiin mechanisms to stop cells from dividing If it didn t since human cells take about a day to duplicate themselves you would be as large as the entire class and weight about 20 tons Mutations can inactivate those growth control mechanisms which would lead to cancer Summary Mutations are rare and unhealthy for the organism but are the driving force behind evolution adaptation to a changing environment M C quot 3 CHAPTER 5 DNA REPLl 3 TION RE AIR AND REC OIVIBINATIQN 36 DNA REPLICATION MECHANISMS DNA replication is fundamental to all organisms All organisms replicate their DNA the same way E coli has been used as the model system Base pamng Underhes DNA Repl1cat1on and DNA Repa1r One strand acts as a template for the other strand Fig 5 2 Remember A pairs with T and C with G Fig 5 3 DNA polymerase is the name of the enzyme that replicates DNA Fig 5 4 The substrates for DNA polymerase are dATP deoxy ATP dTTP dCTP dGTP Collectively these four nucleotide substrates are called dNTPs What s the difference between dATP and ATP How many phosphates does dGTP have Also need are a template and a 3 OH from the growing polynucleotide chain The DNA Replication Fork ls Asymmetrical Replication forks are the point at which DNA replication is occurring Fig 5 6 Replication proceeds in the 5 five prime to 3 three prime direction NOT in the 3 to 5 direction Fig 5 7 Therefore the replication fork must be asymmetric Fig 5 8 Remember that the each strand in the DNA double helix is antiparallel Continuous polymerization in the same direction as the fork is called leading strand synthesis Polymerization in the direction opposite to the fork has to be discontinuous and is called lagging strand synthesis I guess because it takes so long Why must lagging strand synthesis be discontinuous ie synthesized in short stretches What the heck are Okazaki fragments What was the name of the scientist who discovered these fragments You can t just leave them there as fragments How do Okazaki fragments get stitched together rt M 1quot 7 at 37 CHAPTER 5 DNA REPLlCATION REPAIR AND RECOMEUATIGN The High Fidelity of DNA Replication Requires Several Proofreading Mechanlsms Normally base pairing interactions What kind Hydrogen bonding 7 good between the incoming nucleotide base and the template dictate which of the four possible nucleotides is accepted Random nucleotides of all sorts are constantly diffusing in and out of the active site Fig 5 9 Two mechanisms keep the correct nucleotide in the active site long enough to react with the 3 end of the growing polynucleotide chain 1 A and G have big bases C and T have small bases Only a big and small one can occupy the tight quarters of the active site at the same time 2 Proper baseipairing keeps the correct nucleotide there long enough to react Believe it or not the nucleotide bases can morph into other chemical structures This happens very rarely Called tautomerization The morphed structures can fool DNA polymerase For example C can morph into something that pairs with A instead of G After it gets incorporated into the growing polynucleotide chain it can morph back This happens about once for every 100000 nucleotides incorporated If left uncorrected this mutation frequency would be lethal to the cell Before moving on and incorporating the next nucleotide DNA polymerase looks back and checks to see whether the newly incorporated nucleotide is correctly base paired In reality its hard for the growing 3 end to stay in the enzyme active site if it is not properly base paired and thus positioned with the template strand An unpaired nucleotide at the 3 end is apping around in the breeze Fig 5 10 This nucleotide will ap into a different active site located next door to the polymerization active site This adjacent active site cuts off the unpaired nucleotide This enzyme activity is called a 3 to5 proofreading exonuclease About one out of every 100 misincorporations is missed by this proofreading activity The result is continued DNA polymerization with an incorporated mutation Fortunately there is one final check Another enzyme scans the DNA looking for mis matches If it finds one it chops out the mismatched nucleotide more on this later This mismatch repair is able to find 99 out of 100 mismatches If you put together all the proofreading mechanisms nucleotide misincorporation occurs about once every billion incorporation events Table 5 1 r 38 A 5 39 U A m CHAPTER 5 DNA REPLlCATlON REPAIR AND RECOlVlEIAJATION A Special Nucleotide Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand DNA polymerase can only polymerize off of a 3 end Remember it needs an 70H to attack the phosphate on the incoming nucleotide tiiphosphate Why I don t know Perhaps DNA polymerase can t proofread as well on the first nucleotide Anyway the cell initiates DNA replication by first polymerizing RNA on the template instead of DNA Fig 5 12 This RNA is called a primer because it primes DNA synthesis ie gets it going The enzyme is called DNA primase and is a kind of RNA polymerase The RNA is about 10 nucleotides long The RNADNA double helix looks about the same as a DNA double helix except for those two chemical properties unique to RNA Remember what those two properties are On the lagging strand each Okazaki fragment begins with an RNA primer Synthesis of Okazaki fragments initiates at intervals of about every 200 nucleotides along the template How many RNA primers are required for the leading strand Once the RNA primer is made DNA polymerase displaces primase and begins polymerizing DNA Fig 5 13 The RNA is then erased removed by an RNase RNases hydrolyze an RNA polymer into individual nucleotide monophosphates On the lagging strand DNA polymerase polymerizes until it runs into the prior Okazaki fragment The fragments are joined by a DNA ligase Fig 5 14 Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork The DNA double helix is very stable meaning that it is very hard to pull apart the two strands What kind of interactions prevent strand separation Separation of the two strands is called denaturation In order for DNA polymerase to insert new nucleotides the strands have to be separated so that the bases can pair with the correct nucleotide A DNA helicase separates the DNA strands Fig 5 15 5 16 This requires a lot of energy Guess where this energy comes from But the two strands can re anneal or renature same thing to form the double helix again Another problem with single stranded DNA is that it tends to form base pairs with itself particularly if a complementary sequence is nearby Fig 5 17 These intrarnolecular interactions are called hairpins Want to guess why they are called hairpins So what else might be important to keep the strands separated Part lll 39 CHAPTER 5 DNA REPLlCATION REPAIR AND RECUMBINATION A Moving DNA Polymerase Molecule Stays Connected to the DNA by a Sliding Ring After DNA polymerase adds a nucleotide to the growing polynucleotide chain one of two things can happen 1 DNA polymerase can step forward and add another nucleotide 2 Or it can dissociate from the template and diffuse away When would it want to step forward When would it want to dissociate DNA polymerase has the natural tendency to dissociate so a sliding clamp holds it on the DNA Fig 5 19 The sliding clamp forms a ring around the DNA You can see why on a very long piece of DNA how the sliding clamp cannot fall off the DNA So how does the clamp get on the DNA A clamp loader separates the ring allowing it to encircle the DNA Do you think energy is required to do this How is that energy supplied When DNA polymerase runs into the next Okazaki fragment it dissociates Fig 5 20 The Proteins at a Replication Fork Cooperate to Form a Replication Machine So lets review what we have so far Protein Function DNA helicase Separates DNA strands SSB Keeps singleistranded DNA from reiannealing Primase Lays down the RNA primer Clamp loader Loads the sliding clamp onto DNA Sliding clamp Holds the DNA polymerase on the template DNA polymerase Makes the DNA RNase Removes the RNA primer DNA ligase Joins together Okazaki fragments Actually there are a lot more proteins involved but we won t get in to them Many of these proteins work closely together and so they form complexes For example the primase and helicase form a complex called the primosome Actually the whole enchilada is called a replisome Fig 5 22 a 40 REPLlCATlON REPAIR AND RECOl aIEIAATION A Strand directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Machine A protein complex scans newly replicated DNA for bulges in the DNA helix Fig 5 23 You can imagine how a protein can sense a bulging DNA These bulges correspond to mismatches due to incorrect nucleotide incorporation after fooling DNA polymerases proofreading activity The scanning must happen very soon after the new strand is made The complex is called the mismatch repair complex The mismatch repair complex cuts out the mismatched nucleotide and a bunch of surrounding nucleotides as well DNA polymerase then comes along a fills in the gap Big big problem How does the mismatch repair complex know which of the two opposing mismatched nucleotides is the wrongly incorporated the other being the parental template Solution Have the parental strand marked in some way so that the mismatch repair complex know who the parent was How to mark In bacteria methylate the A of a GATC nucleotide sequence of the parental strand In eukaryotes make a break called a nick in the newly synthesized strand Long before replication is initiated a DNA methylase goes along marking the bacterial DNA The newly synthesized strand is unmethylated So the mismatch repair complex finds the unrnethylated strand and cleaves it No problem So there is a race between the mismatch repair complex and the DNA methylase Without the mismatch repair complex you will get more mutations in you DNA and be more susceptible to certain cancers DNA Topoisomerases Prevent DNA Tangling During Replication Strand separation by the DNA helicases causes a topological problem Fig 5 24 The DNA helix ahead of the replication fork get twisted upon itself When a helix twists upon itself this is called a supercoil or superhelical turns An enzyme that removers superhelical twists is called a topoisomerase Some topoisomerases break nick on of the strands of the double helix allowing the other strand to swivel Fig 5 25 Is energy required for this DNA Replication Is Similar in Eucaryotes and Bacteria Summary DNA replication begins by separating the DNA strands Next an RNA primer laid down DNA polymerase initiates off the primer A sliding clamp keeps the DNA polymerase on the template DNA polymerase proceeds continuously on one strand and discontinuously on the other Topoisomerases keep down the supercoiling DNA polymerase proofreads the DNA to make sure it put in the correct nucleotide A mismatch repair complex further proofreads the DNA immediately after DNA polymerase has done its thing 41 Part M CHAPTER 5 DNA REPLICATION REPAIR AND REC OIVIEINATION THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES DNA Synthesis Begins at Replication Origins DNA replication begins at very precise locations Fig 5 29 The replication machinery does not begin at the end of the chromosome or at random locations Why In bacteria there are no chromsomal ends Eukaryotic chromosomes are too long to do enditoiend replication The replication machinery may physically interfere with gene expression Headron collision ofDNA polymerase and RNA polymerase 7 ouch Coordination of the two polymerases could alleviate this problem Certain regions of chromosomes need to replication before other regions A specific sequence of nucleotides comprising the replication origin binds to proteins that specialize in recruiting the replication machinery In bacteria the origin recognition protein is called dnaA protein generically called initiator proteins in the text In eukaryotes the origin is recognized by the Origin Recognition Complex ORC Another feature of replication origins is that part of the sequence has a lot of A and T nucleotides Why Hint 1 AT base pairs are bonded by two hydrogen bonds GAC by three Hint 2 DNA replication requires strand separation Bacterial Chromosomes Have a Single Origin of DNA Replication Fig 5 30 Bacterial DNA replication is regulated at the point of initiation Fig 5 31 Once initiation begins it continues until the whole chromosome is duplicated How do you prevent reinitiation Fig 5 32 Mark the parental strands and only assemble dnaA if both strands are parental DNA methylases chemically modify mark the DNA Eucaryotic Chromosomes Contain Multiple Origins of Replication Eukaryotic chromosomes are large so there is a need for multiple origins of replication A bacterial chromosome has a few million base pairs A eukaryotic chromosome has hundreds of millions of base pairs In Eucaryotes DNA Replication Takes Place During Only One Part of the Cell Cycle There are four phases to the eukaryotic cell cycle growth and duplication of a cell Fig 5 34 39 G1 S G2 M DNA synthesis occurs during S phase a Part llT Meehantsms 42 CHAPTER 5 DNA REPLlCATION REPAIR AND RECOMBINATION Different Regions on the Same Chromosome Replicate at Distinct Times in S Phase Highly Condensed Chromatin Replicates Late While Genes in Less Condensed Chromatin Tend to Replicate Early Well defined DNA Sequences Serve as Replication Origins in a Simple Eucaryote the Budding Yeast A replication origin in yeast is called an ARS autonomously replicating sequence An ARS is a DNA sequence of about 150 base pairs in length and binds multiple protein complexes How might you use the power of genetics to isolate an ARS Fig 5 36 Sequencing of the entire yeast genome has revealed the location of ARSs Fig 5 37 An ARS has binding sites for multiple protein complexes Fig 5 38 A Large Multi subunit Complex Binds to Eucaryotic Origins of Replication This protein complex is called an ORC origin recognition complex An ORC binds to a portion of an ARS An ORC is bound to the chromosome throughout the cell cycle During S phase the ORC is phosphorylated allowing it to recruit other initiation factors helicase etc The l lanimalian DNA Sequences That Specify the Initiation of Replication Have Been Difficult to Identify New Nucleosomes Are Assembled Behind the Replication Fork How does the replication machinery move through nucleosomes Fig 5 41 Remember chromatin remodelling complexes Where do the nucleosomes go after the replication fork has passed How do additional nucleosomes get assembled Chromatin assembly factors CAFs help assemble histones and DNA into a nucleosome r r 43 AIR AND REC OIVIEUATIGN Telomerase Replicates the Ends of Chromosomes How do eukaryotes replicate the ends of their linear chromosomes Fig 5 43 Remember the RNA primer is laid down then removed How do you replicate that part of the chromosome If you don t repeated cell divisions will lead to progressively shortened telomeres Telomerase extends the chromosomal end using an RNA template The RNA template is a component of telomerase Since the template is RNA telomerase is an RNAidependent DNA polymerase aka Reverse Transcriptase Since telomerase repeatedly extends the chromosomal ends eukaryotic chromosomes have repeating sequences at their telomeres A DNA polymerase primase protein complex then performs lagging strand synthesis Note The problem of replicating the actual chromosomal end never actually gets resolved Telomeres are packaged into different protein complexes and have different structures than the rest of the chromosome Telomere Length Is Regulated by Cells and Organisms Somatic cells are initially formed with the full complement of telomeric repeats Telomerase is not made in somatic cells Somatic cells can undergo only a limited number of cell division due to progressive telomere shortening In every cell division the telomere gets a bit shorter After a number of cell division the telomeric repeats are gone and subsequent cell divisions lead to progressive loss of coding information genes located near the telomeres Cells without these genes die called replicative cell senescence This might explain in part why we stop growing It also might provide a mechanism to prevent uncontrolled cell growth cancer Summary DNA replication begins at precise locations on the chromosome called origins Bacteria have one origin eukaryotes have multiple origins An origin recognition complex binds to origins and recruits the replication machinery In eukaryotes DNA synthesis occurs during Sphase of the cell cycle Telomerase is used to maintain the ends of eukaryotic chromosomes Telomerase is a reverse transcriptase that uses RNA to extend chromosomal ends Telomerase is turned off in somatic cells and turned on in cancer cells rt M 1quot 7 a 44 CHAPTER 5 DNA REPLICMION REPAIR AND RECUIVIEIAJATION DNA REPAIR Genetic variability allows a species to evolve and adapt to a changing environment Genetic stability is important for the functioning of an organism Genetic instability leads to cancer aging death and other notisoifun things Table 572 Your body is constantly bombarded by solar radiation nice tan environmental toxins smoke that cigarette and metabolic by products have a big lunch This stuff damages mutates your DNA Mutate means a chemical change in a nucleotide which might alter its coding information Also this stuff can actually break your chromosomes Each cell of your body acquires thousands of mutations a day Fortunately for you you have several potent DNA repair machines lt1 mutation in a thousand escapes these repair machines Without DNA Repair Spontaneous DNA Damage Would Rapidly Change DNA Sequences Mutations can occur at a variety of locations Fig 5 46 Thymidine dimers Fig 5 48 Depurination Fig 5 47 Deamination Fig 5 52 DNA replication prior to DNA repair propagates the mutation Fig 5 49 The DNA Double Helix ls Readily Repaired The beauty of the DNA double helix is that each strand provides an information backup for the other strand Damage one strand and information on the complimentary strand can be used to repair the damage Each and every cell contains many copies of a variety of DNA repair machines DNA Damage Can Be Removed by More Than One Pathway Common features Damage is cut out Nondamaged strand is used as a template to restore the correct nucleotide sequence What enzyme would do this Base excision repair Fig 5 50A Initially just the damage base is removed The enzyme that does this is called a DNA glycosylase Every kind of base mutation has a particular kind of DNA glycosylase designed for its removal The enzyme scans the DNA flipping out each base and checking it for damage Fig 551 Then the sugar phosphate is removed by a different enzyme AP endonuclease Nucleotide excision repair Fig 5 50B Targets large bulky mutations Thymine dimers form UV light Carcinogens in tobacco smoke covalently attach to DNA A long patch of damage DNA is excised 12 nucleotides stretch a 45 REPLlCATlON REPAIR AND RECOl leIAATION The Chemistry of the DNA Bases Facilitates Damage Detection The four bases GATC were selected during evolution in part because deamination does not lead to interconversion to another base Instead they are recognized as noninatural and are removed Double Strand Breaks are Efficiently Repaired Some types of DNA damaging agents break both strand of DNA Fig 5 53 If left unrepaired the chromosomes would fragment A DNA ligase can rejoin the fragments Called nonhomologous endjoining Typically there is a loss of a base The vast majority of the mammalian genome is noncoding so losing a nucleotide is no problem Homologous chromosomes can be used for repair Called homologous endjoining Remember that we get one set of chromosomes from Mom and the other from Dad Cells use the information from one of the chromosomes to repair the other chromosome If the double strand break occurs during the G2 phase of the cell cycle sister chromosomes can be used Cells Can Produce DNA Repair Enzymes in Response to DNA Damage When cells are bombarded by DNA andor protein damaging agents they undergo a stress response Called heat shock response in the textbook since the response pathway was first characterized by a response to high temperatures Stress proteins or heat shock proteins are produce that help stabilize the cell against damage SOS response occurs in response to DNA damage Single stranded DNA is one indicator of DNA damage Results from UV light SOS response is initiated when the recA protein binds to the single stranded DNA This leads the expression of a number of DNA repair genes One of the SOS induced proteins is an error prone DNA polymerase It is used when there is so much DNA damage that the template strand cannot be used to restore the genetic info Because it is damaged too Better to put in any nucleotide and take your chances rather than leave a lethal gap in the DNA DNA Damage Delays Progression of the Cell Cycle When you have damaged DNA the last thing you want to do is duplicate your chromosomes Mutations would get replicated Breaks and singleistranded gaps would be lethal Part of the SOS response is the production of proteins that stop the cell cycle Summary DNA is constantly being damaged by every day living Bases get damaged chemically modified DNA strands get broken Organisms have several DNA repair mechanisms to faithfully maintain their genetic information Ultimately accumulation of unrepaired DNA damage leads to disease and death Part M M CHAPTER 5 DNA REPLlCATIQ 46 a mi r V REPAIR AND RECOMEINATION GENERAL RECOMBINATION The rearrangement or movement of DNA sequences within the genome is called genetic recombination There are two different mechanisms that lead to genetic recombination General or Homologous Recombination Sitespeci c Recombination Recombinant DNA technology is another means of moving DNA around but this only occurs in test tubes being a product human handiwork General Recombination ls Guided by Base pairing Interactions Between Two Homologous DNA Molecules Two chromosomes or any two stretches of DNA are homologous if they are identical or substantially similar in their nucleotide sequence Two homologous pieces of DNA can swap equivalent strands of their double helix The swapped DNA is called a heteroduplex The original parental DNA is called a homodupleX All organisms from bacteria to humans have machinery to conduct homologous recombination The first step in swapping strands is having one strand invade the duplex Called synapsis Of course proper baseipairing drives the proper alignment of the strands Stand swapping then proceeds like a zipper Can occur over thousands of nucleotides Homologous recombination is used to Repair DNA Align chromosomes during meiosis Fig 20 6 20 11 Evolve chromosomes Meiotic Recombination ls Initiated by Double strand DNA Breaks This is homologous recombination that occurs during meiosis described on pp 1130 1139 During meiosis your germ cells egg or sperm must go from a diploid state two sets of chromosomes from Mom and Dad to a haploid state one set when you become a Mom or Dad Each set of chromosomes must align or pair up so that they can segregate to opposite cells The aligning occurs via homologous recombination Fig 5 56 rt M Ila a 47 CHAPTER 5 DNA REPLlCATION REPAIR AND RECOMEUATIGN DNA Hybridization Reactions Provide a Simple Model for the Basepairing Step in General Recombination As a scientist you can artificially swap DNA strands in a test tube Start by having a solution of identical or nearly identical DNA fragments Then separate the strands by heating them to 100 C 7 called melting or denaturation Then cool the them down to let them come back together Called renaturation reanneal or hybridization The two originally paired strands are unlikely to find each other again and are likely to pair with a similar complimentary strand Fig 557 Cells use enzymes to melt out and denature DNA Cells cannot tolerate the conditions used to denature DNA in a test tube DNA helicases separate the strands SSB singleistranded DNA binding protein binds to the separated strands SSB forms a stiff nucleoiprotein structure that allows irrir rnolecular reannealing of two strands but not intrarnolecular reannealing of neighboring sequences called hairpins Some of this should be familiar to you from the DNA replication section The RecA Protein and its Homologs Enable a DNA Single Strand to Pair with a Homologous Region of DNA Double Helix DNA strand exchange is catalyzed by a nucleoprotein filament Fig 5 58 In bacteria the protein component is called RecA in eukaryotes its called RadSl RecARad51 can simultaneously bind single stranded DNA and double stranded DNA to form a three stranded structure Fig 5 59 If the DNAs within the complex are homologous the strands will synapse pair After synapsis the strands exchange Via a zippering action called branch migration RecARadSl use the energy of ATP hydrolysis to promote uni directional branch migration Fig 560 There Are Multiple Homologs of the RecA Protein in Eucaryotes Each Specialized for a Specific Function Also these recombination protein get help from accessory proteins Brcal and Brca2 are one such accessory protein I mention them because defects in these proteins cause meast cancer 48 Part ill C l 39 AND REt i OlleIl JATION CHAPTER 5 DNA RE Ll a General Recombination Often Involves a Holliday Junction Once strand exchange has initiated Via single stranded DNA a reciprocal exchange can occur Fig 5 67 The point at which the strands cross over is called a Holliday junction Fig 5 61 Fig 5 62 The name comes from the guy who discovered them Holliday junctions are recognized branch migrated and cut by a complex of Ruv proteins Fig 5 The cutting7 process is also called resolution or resolved Only two of the strands are cut Two very different outcomes result depending which two strands get cut One is called crossing over where two chromosome arms are exchanged Fig 5 64 The other is called gene conversion where only the strand that formed the heteroduplex is swapped usually thousands of nucleotides Fig 5 66 Selective DNA repair can convert any mismatches to either of the two parental strands General Recombination Can Cause Gene Conversion Summary Homologous recombination allows large stretches of DNA to move from one chromosome to another Homologous recombination involves the swapping of identical or nearly identical strands RecA protein in bacteria or Rad51 in eukaryotes forms a nucleoproteinfilament on DNA RecARad51 aligns DNA molecules using the energy of ATP hydrolysis to compare two DNAs If they base pair they will stay put and RecARad51 continue to exchange strands in a Zippering action Resolution of the crossing over results in chromosomes exchange arms or could result in DNA repair rt III Ila cl 49 CHAPTER 5 DNA REPLICATION REPAIR AND RECUIVIEIIJATION SITESPECIFIC RECOMBINATION In homologous recombination DNA rearrangement required alignment of homologs Strand exchange took place over thousands of nucleotides In site specific recombination strand exchange occurs at a very short specific DNA sequence typically less than 12 base pairs In some cases any sequence will do Long stretches of homology are not required Allows for pieces of DNA to jump around the genome Both sides of mobile genetic element need to be cut Mobile genetic elements can be hundreds to thousands of nucleotides long Examples of mobile genetic elements Transposons aka transposable elements Move around within a cell Generally cannot move between cells unless free DNA is picked up by a cell Viruses eukaryotes and bacteriophages or phages bacteria DNA gets packaged so that it can move between cells About half of the human genome is composed of mobile genetic elements or their relics Relics being elements that have acquired so many mutations that they are no longer capable of moving Sometimes when genetic elements move they take adjacent DNA with them These are mutations that can be detrimental to the cell Transpos1t1onal S1te spec1f1c Recombmauon Can Insert a DNA Element into Any DNA Sequence Genes located on the mobile genetic elements often code for enzymes that aid in the transposition of the DNA Fig 5 69 Transposons encode transposases Tansposons also have genes that code for antibiotic resistance There are three major classes of transposable elements Table 5 3 DNAionly transposons Fig 5770 Some kinds get excised from the chromosome and moved as DNA Others don t get excised but get replicated during the course of movement Retroviralilike transposons These elements first get transcribed into RNA by an RNA polymerase Then reverseitranscribed into DNA The enzyme is called reverse transcriptase Fig 5774 Retroviruses like HIV do this These element encode their own promoters Unlike retroviruses the elements lack genes for coat proteins that would allow them to move from cell to cell Fig 573 Noniretroviral retrotransposons The promoter lies outside of the element and thus is not transposed Uses an RNA intermediate Fig 5776 ill CHAPTER 5 2 m i JWEEE 50 DNA REPL R AND RECOMEINATION DNA only Transposons Move By DNA Breakage and Joining Reactions Some Viruses Use Transpositional Site specific Recombination to Move Themselves into Host Cell Chromosomes Retroviral like Retrotransposons Resemble Retroviruses but Lack a Protein Coat A Large Fraction of the Human Genome Is Composed of Nonretroviral Retrotransposons Different Transposable Elements Predominate in Different Organisms Genome Sequences Reveal the Approximate Times When Transposable Elements Have Moved Conservative Site specific Recombination Can Reversibly Rearrange DNA Fig 5 79 Conservative Site Specific Recombination Can be Used to Turn Genes On or Off Fig 582 Summary Mobile genetic elements move around via site specific recombination Site specific recombination does not require the alignment of homologous chromosomes Recombination occurs at selected anor random locations Some transposable elements move by a cutandpaste mechanism Other transposable elements move via an RNA intermediate Transposable elements primarily move throughout the genome Other mobile genetic elements like viruses are designed to move from cell to cell Some viruses integrate into chromosomal DNA The human genome is mostly composed of repeated sequences that come from mobile genetic elements 3 F g I v we K CHAPTER 6 HC V CELLS READ THE CHAPTER 6 HOW CELLS READ THE GENOME FROM DNA TO PROTEIN JWEEE 51 OME FROM DNA TO PROTEIN s Central dogma DNA makes RNA makes protein Fig 6 2 Bacteria Genes are easy to find Look for a start codon followed by a long gt100 codons open reading frame ORF followed by a stop codon In multicellular eukaryotes the genes are split into small fragments exons separated by long stretches of noncoding DNA introns Process Name DNA 9 pre mRNA Transcription pre mRNA 9 mRNA RNA splicing and 3 cleavagepolyadenylation eukaryotes only mRNA 9 protein TranSlation FROM DNA T0 RNA The more of a certain protein the cell needs the more it transcribes and translates the corresponding gene Fig 6 3 Portions of DNA Sequence Are Transcribed into RNA Transcription is the process by which DNA information is chemically rewritten in a slightly different chemical form One of the strands acts as a template the other does not Fig 6 7 RNA differs chemically from DNA in that it Fig 6 4 uses U instead of T difference of a methyl group has a 2 OH instead ofa 2 H RNA consists of 4 monomer units Fig 6 5 U pairs with A C pairs with G name Pyrimidine Purine name base base uracil U A adenine cytosine C G guanine DNA is almost always a double strand helix RNA is single stranded but forms intramolecular helices that fold upon each other much like proteins do Fig 6 6 Part lll CHAPT ER 6 l 52 Transcr1ptlon Produces RNA Complementary to One Strand of DNA While DNA polymerase makes DNA RNA polymerase makes RNA Fig 6 8 The chemistry of nucleotide polymerization is the same for DNA replication and transcription However the proteins and regulation are very different Only a small portion of the entire genome is transcribed RNA polymerization proceeds in the 5 to 3 direction No primer needed Substrates UTP ATP CTP GTP Transcription can be repeatedly initiated on the same gene Fig 6 9 RNA polymerase exhibits modest proofreading capability Cells Produce Several Types of RNA Table 6 1 mRNA Genes that code for protein are transcribed into mRNA m is for messenger Many different kinds Each with a different range of abundance depending upon cellular needs Eukaryotes one mRNA typically makes one protein Bacteria one mRNA can make multiple proteins Polycistronic mRNA One gene follows another rRNA RNA component of ribosomes ribosomal RNA 90 of all cellular RNA Only a few different kinds tRNA RNAs used to translate codons into amino acids transfer RNA Small typically N75 nucleotides About 30 different kinds all very similar in sequence and structure snRNA small nuclear RNAs that direct RNA splicing snoRNA RNAs that direct chemical modification of rRNA Others telomerase RNA RNAs that inhibit transcription RNAs that inhibit translation Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop The DNA sequence that directs RNA polymerase where to start transcription is called a promoter Every gene has its own promoter RNA polymerase does not directly recognize promoters In bacteria a protein called sigma factor 0 recognizes the promoter and brings in RNA polymerase Fig 6 10 The RNA polymerase then undergoes a conformation change leading to the separations of the DNA stands near the promoter One strand acts a template allowing RNA polymerase to make RNA RNA polymerase moves down the template releasing sigma Eventually RNA polymerase encounter a sequence of nucleotides that causes release of the transcript from RNA polymerase resulting in transcription termination The structure of the RNA polymerase transcribing DNA has recently been determined Fig 611 53 E FROM ow To ROTEIN Transcrlptlon Start and Stop S1gna1s Are Heterogeneous 1n Nucleotlde Sequence When writing the sequence of a gene The coding or transcribed strand is written in a 5339 five prime to three prime direction from left to right The noncoding or template strand is written in the 35539 direction below the coding strand However in the genome not all genes are aligned in the same relative direction The transcription start site is called 1 Do not confuse this with the translation start site which is located within the mRNA transcript Downstream is a term used to imply the direction that RNA polymerase travels RNA polymerase starts at 1 and travels downstream One nucleotide upstream of 1 is called 1 There is no 0 35 nucleotides upstream of the transcriptional start site is called 735 Bacterial promoters are always upstream of the transcriptional start site In bacterial promoters sigma factor binds to a sequence of nucleotides located at 10 and 35 The 10 and 35 region comprise a portion of bacterial promoters The generic term for DNA sequences that bind proteins involved in transcription is promoter element The generic term for proteins involved in transcription is transcription factor The 10 and 35 region of each bacterial promoter are very similar but not necessarily identical Actually there are several different sigma factors Sigma70 070 is the one used at most genes A small number of genes use other kinds of sigma factors each recognizing a different sequence at the 710 and 735 region A consensus sequence is the sequence of nucleotides that are most commonly found at each position of the sequence Thus the o7 consensus sequence for the 710 region is TATAAT The 07 consensus sequence for the 735 region is TTGACA Other proteins that recognize specific sequences will have a different consensus sequence The point is proteins can recognize a variety of related DNA sequences 7 not just one and only one Sequence Some of sequences bind sigma more tightly while others bind sigma weakly Tighter binding corresponds to more frequent recruitment of RNA polymerase and thus more gene expression The concept of promoter elements is applicable to eukaryotes as well Binding sites for hundreds of different transcription factors have been identified Most sites are only 6 8 nucleotides in length and can vary slightly in their actual sequence Simple math indicates that these sites are likely to occur quite often by chance at random locations throughout the genome A siX nucleotide sequence will occur by chance every 46 nucleotides N4000 nucleotides and thus have over a million copies per genome using human as an example There are only 30000760000 human genes So is very difficult to look at the sequence of the genome and predict where promoters will lie n CHAPTER 6 HOW 54 p LnLLS RLAD THE GENOl le FROM DNA TO PROTEIN Transcr1pt1on ln1t1at1on 1n Eucaryotes Requ1res Many Prote1ns Bacteria have only one kind of RNA polymerase Eukaryotes have three kinds of RNA polymerase Table 6 2 RNA polymerase I transcribes rRNA genes RNA polymerase II transcribes mRNA genes RNA polymerase III transcribes tRNA genes All three are structurally similar to each other and to bacterial RNA polymerases Fig 6 15 RNA polymerases I II and 111 have many stably associated subunits 12 Bacterial RNA polymerases have 5 subunits with sigma being one of them RNA Polymerase 11 Requires General Transcription Factors RNA polymerase 11 requires a set of general transcription factors in order to bind to a promoter The general transcription factors for RNA polymerase H are required at all promoters These factors are listed in the table below and are pronounced tee ef two A for TFIIA etc Name DNA of Function recognition site subunits TFIIA none 3 Help H er bind promoters TFHB BRE 1 Links Milli t0 TFH TFIID T AT A box 15 TBP Recognizes promoter and nucleates transcription initiator DPE 14 TAFs complex assembly TFIIE none 4 Helps separate DNA strands lFllF none 4 Links Hall to pol Il TFIIH none 5 Separates DNA strands and phosphorylates pol II How all these proteins arrive at a promoter inside of a cell is not known for certainty When these proteins are isolated from a cell and placed in a test tube containing promoter DNA they will assemble as described in Fig 616 to form a transcription initiation complex Other scientist have isolated nearly the entire complex preiassembled but not yet bound to the promoter The prerassembled complex of RNA polymerase II many of the general transcription factors is called RNA polymerase 11 holoenzyme One important promoter element present at many but not all eukaryotic promoters is the TATA box The TATA box is typically located at 30 not 25as stated in the text It has the following consensus sequence TATAATAATAG note AT means that it can be either A or T The TATA box is recognized by the TATA binding protein TBP Fig 6 18 TBP is a subunit of TFIID Distortion of the DNA by TBP may be important for transcription complex assembly 55 IE FROIVI DNA TO ROTEIN The largest subunit of RNA polymerase two has a repeating amino acid sequence at its carboxy terminal end For the human RNA polymerase II a sequence of seven amino acids is repeated 52 times The carboxy terminal domain or CTD is thought to help tether RNA polymerase II to the general transcription factors and other regulatory factors described below Once the transcription initiation complex has assembled it must separate the two strands at the transcriptional start site just like in bacteria TFIIH has a helicase subunit for this purpose TFIIH kicks off the initiation of RNA synthesis by phosphorylating the CTD of RNA polymerase II An enzyme that phosphorylates another proteinis called a kinase So TFIIH is also a kinase RNA polymerase II is no longer tethered to the other factors at the promoter and is now free to move down the template transcribing RNA Polymerase II Also Requires Activator Mediator and Chromatin modifying Proteins This a very very important point In a test tube also called in vitro RNA polymerase II and the general transcription factors are sufficient to initiate RNA synthesis at a promoter In a cell in vivo the sequences that are recognized by the general transcription factors are necessary but are not sufficient to assembly a transcription initiation complex The reason for this is that there are a lot of things that antagonize transcription complex assembly Huge amounts of nonipromoter DNA compete for transcription factor binding Although binding is weak there is a million times more nonrpromoter DNA than promoter DNA So basic laws of chemistry mass action say that these transcription factors will be tied up at nonrpromoter sites Histones bind DNA to form nucleosomes which can cover up important promoter elements Inhibitor proteins bind to the general transcription factors and prevent them from assembling Transcriptional activators bind to specific DNA sequences and help assemble the transcription complex Fig 6 19 This is so important that the entirety of Chapter 7 is devoted to it Transcriptional activators control gene expression in all forms of life Transcriptional activators help remove nucleosomal inhibitors of promoter elements by recruiting chromatin remodelling complexes Transcriptional activators help remove inhibitors of the general transcription factors Transcriptional activators recruit RNA polymerase II and the general transcription factors through interactions with the following TAF subunits of TFIID TFIIB Mediator Mediator is a multi subunit complex that helps bridge interactions between activators and RNA polymerase II Like the general transcription f actors mediator is required for the expression of nearly all genes Because med39mtor is not strictly required to reconstitute promoterispecific transcription in vitro it was originally missed as a general transcription factor Marina 56 THE GENOME FROM DNA To PROTEIN Transcription Elongation Produces Superhelical Tension in DNA The phase of transcription by which RNA polymerase moves down the template transcribing RNA is called transcription elongation The problem is is that there are a lot of obstacles for RNA polymerase One obstacle in the template sequence itself The RNADNA hybrid needto separate as RNA polymerase moves past RNADNA strand separation is thejob of RNA polymerase Very stable RNADNA hybrids slow the polymerase down Nucleotide misiincorporation slows RNA polymerase down Its difficult to polymerize off of a misrincorporated nucleotide Nucleosomes and other chromatin protein present barriers In all forms of life both RNA and DNA polymerases create superhelical twists in the DNA Fig 6 20 Superhelical twist were discussed earlier in Chapter 5 Superhelical twists impede the movement of the polymerases because the twisted DNA is harder to unwind Topoisomerases relieve this tension and thus enhance elongation Elongation factors help RNA polymerase move through these obstacles True in all forms of life Transcription Elongation in Eucaryotes Is Tightly Coupled To RNA Processing The RNA produced by RNA polymerase II is modified in a number ways Fig 6 21 Fig 6 22a 1 The 5 end of the RNA is capped 2 Introns are spliced out Eukaryotic genes are split into coding exons and noncoding introns Introns are spliced out from the preimRNA or primary mRNA transcript to make the mature mRNA transcript 3 The 3 end of the RNA transcript is cleaved and polyadenylated The enzymes that carry out these reactions might be recruited through interactions with the phosphorylated CTD of RNA polymerase 11 Fig 6 23 Remember the CTD is phosphorylated upon transcription initiation causing initiation factors to dissociate After that the mRNA is exported from the nucleus to the cytoplasm where it is translated Part Til CHAPT ER 6 l 57 RNA Capping Is the First Modification of Eucaryotic Pre mRNAs As soon as the pre mRNA emerges from RNA polymerase II it is capped Fig 6 22b The cap is a modified guanine nucleotide that is added to the 5 end of the transcript Its actually a 5 7toi 5 linkage as opposed to the normal 5 itoi 3 linkage in a standard polynucleotide RNA or DNA chain The cap protects the RNA from being degraded by RNases that recognize unprotected 5 ends 5 caps do NOT exist in bacteria or on transcripts made by RNA polymerase I and III RNA Splicing Removes Intron Sequences from Newly Transcribed Pre mRNAs Eukaryotic genes are split into coding exons and noncoding introns Fig 6 25 Introns are spliced out from the pre mRNA or primary mRNA transcript to make the mature mRNA transcript Exons constitute a very small portion of the genomic sequence of a gene Split genes and RNA splicing is primarily associated with multi cellular eukaryotes RNA splicing involves two sequential transesterification reactions There are different kinds of splicing mechanisms Group II is shown in Fig 6 26 One advantage of having split genes is that different subsets of exons can be spliced together to make different proteins Fig 6 27 So the 30000 different human genes may actually code for gt100000 different proteins Nucleotide Sequences Signal Where Splicing Occurs As with everything else the location of the splice sites is determined by the nucleotide sequence at the splice sites as well as a sequence element located internal to the intron Fig 628 Intron range in size from 10 t010000 nucleotides RNA Splicing ls Performed by the Spliceosome snRNA are used to direct the splicing reaction The Spliceosome is a large RNAprotein complex that conducts pre mRNA splicing Fig 6 29 The Spliceosome is composed of several snRNPs snurps and each snRNP is composed of an snRNA U1 snRNP U1 snRNA splicing proteins U2 snRNP U2 snRNA splicing proteins U4 snRNP U4 snRNA splicing proteins U5 snRNP U5 snRNA splicing proteins U6 snRNP U6 snRNA splicing proteins snRNAs recognize the splice sites through base pairing interactions Fig 6 30 rt llT s a a 58 CHAPTER 6 HOW CELLS READ THE GENOlVlE PROM DNA TO ROTEIN The Spliceosome Uses ATP Hydrolysis to Produce a Complex Series of RNA RNA Rearrangements Some of the splicing proteins are RNA helicases which use the energy of ATP hydrolysis rearrange RNA RNA interactions Fig 6 30 The active site for the transesterification reaction of splicing are made up of RNA Spliceosome catalyzed RNA Splicing Probably Evolved from Self splicing Mechan1sms In lower eukaryotes certain splicing reactions are autocatalytic requiring no protein There are two different kinds of self splicing reactions Fig 6 36 Group I The RNA intron forms a tertiary structure that binds to a free guanine G nucleotide cofactor in its active site The 3 70H of the free G initiates the first transesterification reaction Group II The RNA intron forms a tertiary structure that positions an adenosine nucleotide in the active site The adenosine A nucleotide is part of the intron sequence 1 call it the intronic A The 2 70H of the intronic A initiates the first transesterification reaction forming a lariat structure A lariat has both 25539 and 35539 phosphodiester bonds Splicing of pre mRNA transcribed by RNA polymerase 11 using the spliceosome evolved from the group II mechanisms to include the use of protein and non intronic RNA ie snRNA probably to assist in the regulation of splicing and alternative splice site selection Since the intron sequence was no longer required to provide the tertiary structure intron sequence rapidly diverged Less constraints on intronic structure also allowed the number of introns and exons to proliferate It is interesting that the complex human beings don t have much more genes than worms flies or plants But they do have the capacity to produce many more proteins though the use of additional exons rt M s a a 59 CHAPT ER 6 HOW CELLS READ THE GENOlVlE FROM DNA TO ROTEIN RNA Processing Enzymes Generate the 3 End of Eucaryotic mRNAs mRNA that is not protected is rapidly degraded by RNases RNases can chew in from the 5 and 3 ends The guanosine cap protects the 5 end The 3 end is protected by polyadenylation Fig 6 37 Both reactions are specific to transcripts made by RNA polymerase II First the mRNA is cleaved Fig 6 38 As RNA polymerase II transcribes through the end of a gene it encounters the sequence AAUAAA I call this sequence A2UA3 A multiisubunit cleavage complex that loaded onto RNA polymerase II CTD via TFIID scans the emerging RNA and binds to the A2UA3 sequence The nascent newly made RNA is then cleaved about 30 nucleotides downstream of the A2UA3 sequence Second a polyA polymerase associates with the 3 end of the mRNA transcript and polyadenylates it The polyA polymerase is a templateiindependent RNA polymerase that uses only ATP as its substrate Approximate 200 A nucleotides are added PolyA binding proteins bind to this polyA tail forming an RNAprotein complex Binding of the polyA binding protein regulates polyA polymerase A protein RNA structure that accommodates N200 A s is probably the most stable thus limiting the amount of polyadenylation Mature Eucaryotic mRNAs Are Selectively Exported from the Nucleus RNA processing of pre mRNA occurs in the nucleus Most of the RNA in the nucleus is the discarded introns which are selectively degraded Probably because they lack a cap and are not polyadenylated and thus are susceptible to RNase attack Also introns remain bound to a class of proteins that form an hnRNP Lheterogeneous r1uclear ribonuclear protein complex So named because they were varied in size as View under the electron microscope The proteins that are bound to the mRNA s cap exons and polyA tail are recognized by the cells export machinery The RNA is transported through the nuclear envelope into the cytoplasm via nuclear pores Fig 6 39 Nuclear pores are aqueous channels lined with protein that traverse the nuclear envelope As the mRNA traverses the nuclear envelope many of the associated proteins are replaced with cytosolic counterparts Fig 6 40 The cytosolic factors protect the mRNA from degradation and aid in the assembly of the ribosome The ribosome is where the mRNA is translated itleeihai 60 THE GENOME FROM DNA To PROTEIN 0K lets take a break from the exciting travails of the mRNA journey so that we canfind out more about what other nonmRNAs do Many Noncoding RNAs Are Also Synthesized and Processed in the Nucleus Ribosomal RNA rRNA is made in the nucleolus by RNA polymerase 1 Most of the RNA in a cell is rRNA The nucleolus is described below RNA polymerase I lacks the CTD found on pol II and so rRNA is not processed in the same way as pol II transcripts NO cap NO splicing NO polyadenylation Nada The ribosome contains one copy of 4 different kinds of rRNA Fig 6 42 Three of the four are made by cutting up a pre rRNA transcript Remember this is rRNA cutting and not mRNA splicing Their names are 188 588 and 288 Whichis a reflection of their size snoRNPs direct the cutting up of the preirRNA They are NOT the same kind of snRNPs involved in splicing The fourth called SS rRNA is synthesized by RNA polymerase III outside of the nucleolus The rRNAs are methylated at their 2 OH Fig 6 43 Only certain rRNA nucleotides get methylated snoRNAs are small nucleolar RNAs that bind to specific rRNA sequences providing a guide for a methylase Purpose aid in folding stability from degradation As an interesting twist snoRNAs are often derived from introns of genes encoding ribosomal proteins The Nucleolus Is a Ribosome Producing Factory The nucleolus is a substructure within the nucleus Fig 6 44 It is where ribosomes are made It is formed from the coalescence of rRNA genes RNA polymerase I rRNA tRNA ribosomal proteins snoRNPs and partially assembled ribosomes Summary Proteins and RNA do all the work in the cell making us who we are Have you thanked your proteins and RNA today The instructions for making protein and RNA is encoded in genes located on chromosomes RNA polymerase and other help proteins recognize specific sequences of nucleotides that demarcate the beginning of the gene called a promoter RNA polymerase transcribes the genes into RNA The genes of multicellular genes are composed for coding exons and noncoding introns that are spliced out and generally discarded A spliceosome is an RNAprotein complex that splices out introns Eukaryotic mRNAs are modified having a guanosine cap Q at its 5 end and apoly A tail Elgat its 3 end Processed mRNA are exported to the cytoplasm where they are translated NonmRNAs such as snRNA assist with a nuclear functions including RNA splicing The nucleolus is where rRNA is made modified and packaged intro ribosomes 61 GENOA1E FROM DNA TO ROTEIN FROM RNA TO PROTEIN An mRNA Sequence Is Decoded in Sets of Three Nucleotides The genetic code posits that the sequence of three nucleotides codon is the code word for a particular amino acid Fig 6 50 The nucleotide sequence of mRNA is read as a series of codons There is a one to one correspondence between a codon and an amino acid Fig 6 51 Actually there are three codons that signify translation to stop 7 called stop codons The codon for the amino acid methionine AUG is where translation starts 7 called the start codon An open reading frame ORF is a series of codons typically gt75 that are bounded by a start codon and stop codon when read the mRNA is read in the 373 direction All forms of life use the same genetic code One notable exception mitochondria have there own genome and translation machinery so the genetic code differs slightly 7 but still 3 nucleotides to a codon Since a codon has three nucleotides an RNA has three theoretical reading frames of which only one is used There are 4x4x464 possible codons of which 61 code for 20 different amino acids Obviously some amino acids are specified by more than one codon The other 3 codons are stop codons The mRNA is translated by a ribosome tRNA Molecules Match Amino Acids to Codons in mRNA A codon does not directly match up to an amino acid A particular amino acid is covalently attached to a particular tRNA Fig 6 52 Since there are 61 different codons for 20 amino acids are there 61 different tRNAs No The number is somewhere between 20 and 61 depending upon the organism Reason Some tRNAs can recognize more than one kind of codon Fig 6 53 These tRNAs require only proper base7pairing at the first two nucleotides of the codon Historically called a wobble So the code is often degenerate at the third position For example GGA GGC GGG and GGU all code for glycine A 3 nucleotide sequence in the tRNA called an anticodon pairs with the appropriate codon What type of interactions are responsible for the pairing tRNAs Are Covalently Modified Before They Exit from the Nucleus Eukaryotic tRNAs are made by RNA polymerase 111 Some contain introns that are spliced out by mechanisms totally unrelated to mRNA splicing a quot litleelnan sms 62 E Ls READ THE GENOMfE FROM DNA To PROTEIN Fart lll CHAPTER 6 HOW C Specific Enzymes Couple Each Amino Acid to Its Appropriate tRNA Molecule Aminoacyl tRNA synthetases covalently attach the proper amino acid to the 3 end of the tRNA Fig 656 657 Every type of amino acid has its own kind of aminoacyl tRNA synthetase So there are 20 different but structurally similar aminoacylitRNA synthetase Editing by RNA Synthetases Ensures Accuracy It is critical that the correct amino acid be attached to the correct tRNA Aminoacyl tRNA synthetases employ editing mechanisms analogous to that of DNA polymerase during DNA replication Fig 6 59 If the correct amino acid is not covalently attached it is removed Aminoacyl tRNA synthetases recognize the correct tRNA through interactions with the anticodon Fig 6 60 Amino Acids Are Added to the C terminal End of a Growing Polypeptide Chain Like polynucleotide chains amino acids and polypeptides have a directionality Fig 6 61 The polypeptide chain is synthesize in the N terminal to C terminal direction Every new amino acid is added at the Citerminal end Part M CHAPT ER 6 l 63 The RNA Message ls Decoded on Ribosomes Fig 6 68 Please marvelal its beauty The ribosome is an RNA machine that is decorated with a bit of protein It contains a large subunit and a small subunit each of which is composed of many subunits Fig 6 63 Bacterial and eukaryotic ribosomes are very similar A single mRNA can be translated simultaneously by many ribosomes The small subunit functions to pair the tRNA with the mRNA The large subunit catalyzes the peptidyltransferase reaction protein synthesis The small and large subunit associate on the mRNA near the 5 end with the small subunit entering first The ribosome moves down the mRNA recruiting the appropriate aminoacylated tRNA and synthesizing the polypeptide A ribosome can do 20 addition per second in bacteria There are three adjacent tRNA binding sites each with a different function Fig 664 There are three major steps to each addition of an amino acid Fig 6 65 1 The middle P site is bound by a tRNA having the growing polypeptide chain covalently attached to its 3 end The adjacent A site receives a new amino acylated tRNA as directed by the codonanticodon interactions 2 The amino group of the amino acid in the A site is adjacent to the aminoacyl bond of the C terminus of the growing polypeptide The amino group attacks the unstable protein tRNA ester linkage transferring the polypeptide from the P site to the A site But the large subunit quickly moves forward placing the two tRNAs into the E and P s1tes This central activity of the ribosome is called peptidyltransferase 3 Using the energy of GTP hydrolysis the small subunit translocates 1 codon downstream When a stop codon is encounter a release factor that looks like a tRNA but is really protein enters and leads to peptide release Elongation Factors Drive Translation Forward Fig 6 66 EF Tu delivers the tRNAs to the ribosome using the energy of GTP hydrolysis Accuracy is achieved by allowing the amino acylated tRNA to dwell in the A site before it is allowed to attack the polypeptide chain Improperly paired tRNAs dissociate more quickly than properly paired ones Because anticodoncodon baseipairing is what holds the tRNA in the A site EF G hydrolyze GTP to power the forward movement of the ribosome large subunit Part III CHAPTER 6 l 64 The Ribosome Is a Ribozyme Fig 6 68 illustrates that the meat of the ribosome is RNA In fact if all the proteins are removed artificially in a test tube the RNA still has peptidyltransferase activity The proteins seem to help hold the ribosome together and assist in some of its conformational changes RNA molecules that catalyze chemical reactions are called ribozymes What other ribozymes have we heard of Fig 670 Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis Where on the mRNA does the ribosome know to begin translation The translation start site sets the reading frame Not to be confused with the transcription start site Translation always begins at an AUG For Eukaryotes Fig 6 71 A methionine charged initiator tRNA is delivered to a free ribosomal small subunit through interactions with a protein complex called eIF Z ukaryotic initiation factor 2 This complex then recognizes the 5 end of the mRNA through interaction with factors eIF 4E and eIF 4G bound to the mRNA guanosine cap Using the energy of ATP hydrolysis the small subunit and its entourage move along the mRNA Once the first AUG is found via anticodoncodon interactions with the met tRNA everything stop and waits until the big guy large subunit comes along Now translation proceeds as described above For Bacteria Fig 6 72 Same sort of thing as for eukaryotes except there is no cap complex to recognize The rRNA in small ribosomal subunit base pairs with an RNA sequence called Shine Dalgarno sequence just upstream of the AUG start codon So the Shine Dalgarno sequence directs where translation begins in bacterial mRNAs This allows for internal starts as would be necessary for polycistronic messages Stop Codons Mark the End of Translation There is no tRNA that is designed to bind to one of the three stop codons Instead a protein called eRF eukaryotic release factor binds Fig 6 73 eRF looks very much like a tRNA Fig 6 74 Called molecular mimicry Instead of adding an amino acid to the growing polypeptide chain as tRNAs do eRF adds H20 The polypeptide is now no longer attached to anything and so it diffuses away rim quot 65 ELLE Rn D THE EN JME PROM DNA To PROTEIN Proteins Are Made on Polyribosomes Once a ribosome starts translating another ribosome can start right behind it Fig 6 75 eIF 4G also binds to the polyA binding protein making the 5 and 3 end of the mRNA right next to each other A Protein Begins to Fold While It Is Still Being Synthesized As the N terminal end emerges from the ribosome it starts to adopt secondary structure alpha helices and beta sheets Tertiary interaction involving amino acid side chains start to happen but these interactions constantly rearrange settling on the most stable set of interactions before the final structure is obtained Fig 6 80 Protein domains fold sequentially Fig 6 81 Molecular Chaperones Help Guide the Folding of Many Proteins While proteins can fold into their final 3 D structure by themselves other proteins called molecular chaperones make the folding process more efficient Fig 6 82 Molecular chaperones are also important for helping cells deal with stress such as high heat Heat can cause proteins to misfold or not fold Many molecular chaperones are also heat shock proteins hsp Different molecular chaperones work in different ways Fig 6 84 Exposed Hydrophobic Regions Provide Critical Signals for Protein Quality Control Hydrophobic regions of the protein do not want to be exposed to water So they normally bury themselves deep within the protein Misfolded proteins tend to have a hydrophobic patches on their surface These hydrophobic patches are recognized by chaperones If chaperones are unable to help refold the protein the misfolded protein is ultimately degraded recycled by proteases Proteases are enzymes that cut proteins into small peptides Part lll CHAPTER 6 l 66 The Proteasome Degrades a Substantial Fraction of the Newly Synthesized Proteins in Cells A proteosome is a large multi subunit complex that degrades proteins Fig 6 86 It includes enzymes with protease activity An Elaborate Ubiquitin conjugating System Marks Proteins for Destruction Ubiquitin is a short protein Surveillance proteins recognize damaged and misfolded proteins These protein covalently attach ubiquitin onto the targeted protein The proteosome recognizes the ubiquitin and degrades anything bound to it Many Proteins Are Controlled by Regulated Destruction Many proteins need to be rapidly disposed of in response to a changing cellular environment For example as a cell goes through the cell cycle certain protein must be rapidly removed This is done by regulated degradation Proteins can have a degradation signal that is hidden Fig 6 88b Protein phosphorylation dissociation of a subunit or cleavage of the protein can expose its degradation signal Abnormally Folded Proteins Can Aggregate to Cause Destructive Human Diseases Excessive misfolding of proteins can lead to their aggregation Aggregated proteins can cause diseases such as Alzheimer s and Mad Cow disease There Are Many Steps From DNA to Protein Each step can be regulated turned on or off by cellular signals Summary mRNAs contain a series of threenucleotide codons which get translated into protein Each codon corresponds to a particular amino acid mRNA is recognized by the ribosome located in the cytoplasm Proteins are synthesized on ribosomes tRNAs attached to specific amino acids recognize each codon The codons align the tRNAs allowing the amino acids to polymerize As the polypeptide chain emerges from the ribosome it starts to fold forming its final 3D structure Chaperones are proteins that help other proteins fold properly Misfolded proteins or proteins bearing degradation signals are rapidly degraded in the proteosome All these step from the gene to the lifeandtimes of the protein are regulated by cellular signals Part 11 Basic Genetic Mechanisms 67 CHAPTER 6 HOW CELLS READ THE GENOME EROM DNA TO PROTEIN THE RNA WORLD AND THE ORIGINS OF LIFE RNA probably existed long before DNA This is a very interesting section of the text but unfortunately we do not have time to cover it in class Please read it if you are interested in current theories about how life came into existence llI CHAPTER7 3 CHAPTER 7 CONTROL OF GENE EXPRESSION owns 68 am AN OVERVIEW OF GENE CONTROL The Different Cell Types of a Multicellular Organism Contain the Same DNA Nearly all cells in an organism contain the same set of genes Animals have been cloned by placing the nucleus of a skin cell into an enucleated egg Different Cell Types Synthesize Different Sets of Proteins Cells within the same organism become different because they express a different set of genes Fundamentals about cell differentiation 1 All cells have a set of proteins that are the same eg ribosomal proteins cytoskeleton RNA polymerase etc 2 Specialized cells have certain proteins that are highly abundant eg hemoglobin in red blood cells and not found in other cells 3 Every type of cell has a characteristic pattern and intensity of gene expression Fig 4 This results in a characteristic pattern of protein production A Cell Can Change the Expression of Its Genes in Response to External Signals Cells sense chemical signals from other cells or from the environment These signals elicit specific changes in gene expression Different types of cells might respond differently to the same signal Signal Responding Response cell type glucocorticoid liver increase expression of the tyrosine aminotransferase gene as hormone well as change expression of certain other genes glucocorticoid fat cell decrease expression of the tyrosine aminotransferase gene as hormone well as change expression of certain other genes Part lll 69 CHAPTER 7 CONTROL or GENE EXPREssroN Gene Expression Can Be Regulated at Many of the Steps in the Pathway from DNA to RNA to Protein Points at which the activity of a protein or its gene can be regulated Gene expression RNA processing capping splicing polyadenylation Subcellular localization of mRNA Translation Degradation of mRNA Modification of the protein covalent modification proteinsmall molecule ligand binding Subcellular localization of the protein Destruction of the protein Summary The genome of a cell can make a variety of cell types A cell type is determined by its compliment of RNAS and protein The production and activity of RNAsproteins is highly regulated by cellularenvironmental signals 70 M C quot r 3 JroN CHAPT ER 7 CONTROL L 3 GENE EXPR DNABINDING MOTIFS IN GENE REGULATORY PROTEINS How does the cell selectively turn on or off genes The Outside of the DNA Helix Can Be Read by Proteins The DNA double helix has a minor and major groove Fig 7 6 Each of the four nucleotide bases presents a unique arrangement of hydrogen bond donor and acceptors and hydrophobic patches in the major groove Fig 7 7 A series of nucleotides can present a unique docking site for a protein with a complementary interacting surface Fig 7 8 Table 7 1 Sequences are often 6720 base pairs Short DNA Sequences Are Fundamental Components of Genetic Switches Gene Regulatory Proteins Contain Structural Motifs That Can Read DNA Sequences Protein DNA interactions just like any other interaction depends upon an exact fit Fig 7 12 The 3 D surface of the protein is shaped to accommodate the binding of a DNA double helix Amino acid side chains make precise hydrogen bond interactions with the bases Hydrophobic and van der Waals interactions also take place Ionic interaction take place between amino acids and the DNA phosphates Interaction with the sugar or phosphates generally increase affinity and not speci city Interactions with the bases provide both affinity and specificity Specificity is the ability to discriminate one DNA sequence from another You must know the difference between affinity and specificity 71 The Helix Turn Helix Motif Is One of the Simplest and Most Common DNA binding Motifs Helixturnhelix is a protein motif designed to fit into the major groove Fig 7 13 Composed of two alpha helices Many different gene regulatory proteins have a helix turn helix motif Fig 7 14 By changing some of the amino acids on its DNA recognition helix you can change the DNA binding specificity of the protein A feature common to many sequence specific DNA binding proteins is that they bind DNA as dimers A dimer is a protein complex having two subunit which many times are identical The helixiturnihelix motif on each monomer is spaced apart at the same distance as one turn of the DNA double helix Proteins that bind DNA as dimers must recognize a symmetrical DNA sequence Fig 7 16 Homeodomain Proteins Constitute a Special Class of Helix Turn Helix Proteins Homeodomains are DNA binding motifs found in gene regulatory proteins that direct eukaryotic development That s what makes them so special Mutation in the homeodomain can cause severe developmental problems For example a fly antennae might turn into a leg There Are Several Types of DNA binding Zinc Finger Motifs Another general group of DNA binding motifs use zinc to hold their structure together One member of the group is called a zinc finger Fig 7 17 Several zinc fingers are strung together to form a DNA binding domain Fig 7 18 Each zinc finger sticks an alpha helix into the major groove to achieve binding specificity Fig 7 19 The Leucine Zipper Motif Mediates Both DNA Binding and Protein Dimerization Some proteins dimerize via interaction of two alpha helices Fig 7 21 3 11 Called a leucine zipper So named because hydrophobic leucine sideichains protrude from each alpha helix and interdigitate like a zipper Beyond the interacting helices the helices extend and bifurcate outward allowing them to bind the DNA major groove Think of a scissor grip 72 Heterodimerization Expands the Repertoire of DNA Sequences Recogn1zed by Gene Regulatory Prote1ns A homodimer is a dimer with identical subunits A heterodimer has two different subunits Two different leucine zipper containing proteins can form a heterodimer Fig 7 22 Since they have different DNA binding specificities the heterodimer can bind sequences that neither homodimer could and visa versa Thus fewer proteins can be used to generate a larger DNA binding repertoire Amino acids around the leucines provide specificity and affinity whereas the leucines provide affinity for dimerization The Helix Loop Helix Motif Also Mediates Dimerization and DNA Binding Don t confuse these with helix turn helix proteins Fig 7 25 Helix loop helix proteins are more like leucine zipper proteins A helix loop helix protein could heterodimerize with a different one that lacks a DNA binding domain The complex won t be able to bind DNA Example of negative regulation of the geneispecific regulator Read the next five sections if you plan to work in a laboratory that studies gene regulation Summary Proteins recognize specific DNA sequences through precise docking of amino acid side chains on the protein on to the DNA major groove Specificity is provided through hydrogen bond interactions between the DNA bases and the protein amino acid side chains Many sequencespecific DNA binding proteins use similar structural motifs Sequencespecific DNA binding proteins regulate the expression of a gene 73 HOW GENETIC SWITCHES WORK Above we had a look at gene regulatory proteins Now we look at how they regulate gene expression The Tryptophan Repressor Is a Simple Switch That Turns Genes On and Off in Bacteria This is about as simple as it gets E coli has about 4000 genes 5 of them are dedicated to the synthesis of the amino acid tryptophan All 5 genes are located within the tryptophan operon Fig 7 33 All genes within an operon are transcribed onto a single mRNA The production of that mRNA is controlled by a single promoter E coli lives in your intestines When you eat a hamburger the protein is digested to amino acids including tryptophan So the tryptophan operon can be turned off How Tryptophan binds to a site on the tryptophan trip for short repressor causing the repressor to rotate its DNA recognition helices such that it can now recognize a specific sequence on the tryptophan promoter Fig 7 35 The tryptophan repressor bound to the promoter prevents RNA polymerase from binding Result operon is shut off Fig 7 34 This is an example of negative control or negative regulation In this case a ligand tryptophan caused a repressor to bind DNA In other cases a ligand could cause a repressor to dissociate from DNA Fig 7 36a In this case the ligand is an inducer gene expression Transcr1pt1onal Act1vators Tum Genes On Bacterial RNA polymerases can recognize promoters through interactions of its sigma subunit with specific promoter sequences Many bacterial promoters have suboptimal binding sites for sigma and thus have an intrinsically low capacity to recruit RNA polymerase and transcribe the gene Promoters are designed this way so that RNA polymerase does not continually transcribed the gene Continual transcription gene is wasteful if note needed and could be harmful Its better to have the gene off in the default state Bacteria use regulatable sequence specific binding proteins to recruit and stabilize the binding of RNA polymerase at the promoter The generic term for this type of protein is transcriptional activator The type of regulation is called positive regulation or positive control As with negative control a ligand can help or hinder the binding of transcriptional activator to a promoter Fig 7 36b Some gene regulatory proteins can act as both an activator or repressor depending upon where its DNA binding site is located Fig 7 38 7 37 74 A Transcriptional Activator and a Transcriptional Repressor Control the lac Operon Rationale for regulation E coli normally eat glucose to provide carbon and energy When no glucose is around in the environment they look around for other kinds of sugars e g lactose But only make the lactose metabolizing enzymes if lactose is present Al orithm Algorithm Mechanism Fig 7 38 If glucose and lactose then turn on off CAP bound lac repressor is is the lac operon to promoter bound to promoter present present off No No present absent off No Yes absent absent off Yes Yes absent present on Yes No Mechanism Lac repressor binds to promoter and blocks RNA polymerase binding Lac repressor allolactose ligand cannot bind to promoter CAP CAMP ligand bind to promoter and recruits RNA polymerase Absence of glucose causes cAMP levels to rise Gene regulation in bacteria can be thought of as a series of genetic on off switches Regulation of Transcription in Eucaryotic Cells Is Complex The complexity of eukaryotic cells requires something more than just simple onoff switches Features of eukaryotic regulation 39 Regulatory proteins can be targeted to sequences located thousands base pairs upstream or downstream of the transcription start site 39 Multiple components must come together at a promoter RNA polymerase general transcription factors mediator etc 39 Transcriptional start sites can be covered by histones chromatin Eucaryotic Gene Regulatory Proteins Control Gene Expression from a Distance DNA in a cell is compacted So a protein bound at a distant site might be physically close to the transcription start site Part lll 75 CHAPTER 7 CONTROL OF GENE EXPREssroN A Eucaryotic Gene Control Region Consists of a Promoter Plus Regulatory DNA Sequences A eukaryotic promoter is considered to be the DNA sequence near the transcriptional start site that is required for the binding of the general transcription machinery However keep in mind that in almost all cases the promoter is insufficient to bind the transcription machinery inside of a cell Transcription complex assembly requires gene regulatory proteins bound to DNA regulatory sites Fig 7 41 Different names often refer to the same thing DNA Proteinquot 39 regulatory site 39 activators 39 regulatory sequence 39 transcriptional activators 39 enhancer 39 transcription factors 39 activator binding sites 39 sequence specific activators 39 UAS in yeast upstream activating sequence 39 gene regulatory proteins 39 transcription factor binding sites 39 gene activator proteins 39 DNA binding elements 39 enhancer binding proteins 39 trans activators Some of these terms refer only to positively acting factors other refer to both positive and negative factors There are a few hundred of different kinds of gene regulatory proteins Each target an overlapping set of genes Any one gene mightbe regulated by only a few of them So any one of them might not be present in a particular cell type If they are present they are not abundant Eucaryotic Gene Activator Proteins Promote the Assembly of RNA Polymerase and the General Transcription Factors at the Startpoint of Transcription Transcriptional activators have a modular design Fig 7 42 Transcriptional activators have a DNA binding domain Transcriptional activators have an activation domain Activation domain stimulate transcription These two different types of domains can be mixed andmatched artificially Activation domains plug into the general transcription machinery andor mediator Fig 7 43 744 Activation domains help localize and stabilize the binding of the general transcription machinery to the promoter 76 N Eucaryotic Gene Activator Proteins Modify Local Chromatin Structure Activators also recruit chromatin remodelling factors Fig 7 45 Some remodelling complexes chemically modify the histones Others rearrange the histones on the DNA The remodeled chromatin may allow more ready access of the transcription machinery to the DNA Fig 7 45 Gene Activator Proteins Work Synergistically Transcriptional synergism occurs when single activators don t stimulate transcription but multiple activator do Fig 7 47 Synergism can arise when activators function to accelerate more than one step in transcription initiation Fig 7 48 Eucaryotic Gene Repressor Proteins Can Inhibit Transcription in Various Ways Fig 7 49 Block activator binding to DNA Mask activation domain Mask activator target in the general transcription machinery Reposition nucleosomes over promoter Remove chemical modification of histone that allow factor access Eucaryotic Gene Regulatory Proteins Often Assemble into Complexes on DNA In reality eukaryotic gene regulation is a continuous and dynamic interaction of a variety of positively and negatively acting regulatory proteins Cellular and environmental signals tip the posneg balance in one direction or the other to turn up or turn down the expression of a gene There are many protein involved some bind DNA others do not Fig 7 50
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