Bsci105, review guide for test 3
Bsci105, review guide for test 3 BSCI105
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Chapter 10- Photosynthesis Preview- - Photosynthesis in a nutshell- chloroplasts of plants capture light energy and convert it to chemical energy that is stored in sugar and other organic molecules. – - Nourishes entire world o Organism gets organic compound it uses for energy and carbon skeleton by one of 2 ways Autotrophs- self feeders- sustain themselves without eating other things that are derived from other living things, but can be a source for others. Produce their organic molecules from CO and other 2 raw inorganic materials from the environment Plants are because they just get water and nutrients from the soil and CO fro2 air- specifically photoautotrophs because they use light also to synthesize organic molecules Heterotrophs- get organic compounds from other organisms; consumers Animals eat other living things Dependent on photoautotrophs for food and O 2 (byproduct of photosynthesis) Photosynthesis probably originated from bacteria’s molecules in plasma membrane, which is now known as a chloroplast in a eukaryotic cell. Chloroplasts- site of photosynthesis - Make the plant green from chlorophyll (in the thylakoid) and found mostly in mesophyll- leaf’s interior tissue o CO en2ers leaf and O exits2by pores called stomata, and water is absorbed through veins of leaf - Stroma- liquid in chloroplast, thylakoids- separates the stroma from the thylakoid space within these sacs, each stack of sacs is called granum. - Light energy absorbed by chlorophyll drives the synthesis of the organic molecules Process of Photosynthesis- - 6CO +212H O +2light energy yields C H O + 66 12 6 2+ 6H 2 - Opposite of cellular respiration - O 2iven off from plants is derived from H O, no2 CO - chlor2plast splits the water into H and oxygen Redox Reaction- - 6CO (2xidizing agent) becomes reduced into C H O 6 12 6 - 6H 2 (reducing agent) is oxidized to 6O 2 - Electrons increase potential energy as they move from water to sugar- it requires energy, which means it is endergonic, and this energy is provided through light Preview of Photosynthesis - It is two processes- light reactions (photo part) and Calvin cycle (synthesis part) - Light reactions- water is split, providing H ions and giving of O a2 a by- product. Light absorbed by chlorophyll causes transfer of electrons and H ions to NADP+ (acceptor- extra phosphate in this compared to NAD+). Use solar power to reduce NADP+ to NADPH by adding a pair of electrons with H+. generate ATP by photophosphorylation (adding a P to ADP)- light energy is now converted to chemical energy in form of NADPH and ATP o Occurs in thylakoids o On outside of thylakoids, NADP+ and ADP pick up electrons and phosphate, and then NADPH and ATP are released into stroma - Calvin cycle (can be referred to as dark reaction- don’t need light)- CO 2 from air into organic molecules present in chloroplast (carbon fixation). Reduces fixed carbon to carbohydrate by addition of electrons, and this reducing power is provided by NADPH. The ATP and NADPH is needed for the synthesis to occur. o Occurs in stroma Nature of Sunlight- - Light is electromagnetic energy that travels in waves o Distance between the two crests is called wavelength (from less than a nm to more than a km) and the entire range is known as electromagnetic spectrum o Visible light- between 380nm and 750 nm- can be recognized o Photons- discrete particles in light, the shorter the wavelength, the greater energy of each photon - Pigment- substances that absorb visible light and each absorb different wavelength o If it absorbs all colors, it appears black. If it reflects all colors, it appears white. o The color you see is reflected back, while the pigment absorbs the shorter wavelengths o Light can perform work in chloroplasts only if it is absorbed there What happens when chlorophyll and other pigments absorb light? - is elevated to an orbital where it has more potential energy, which went from ground state Colors with absorbed wavelengths disappear, but energy cannot disappear. When a molecule absorbs a photon, one molecules electrons to excited state. (the only photons absorbed are those whose energy is exactly equal to the energy difference between ground and excited state, making each pigment have a unique absorption spectrum) - The electron can’t remain in excited state for long since it is unstable and generally drop right back down to ground state, releasing excess energy as heat/photons. o When photons are given off, there is an afterglow (fluorescent) Photosystem- - A reaction center complex associated with light harvesting complexes, Chlorophyll and other organic molecules are organized into a photosystem, converts light energy into chemical energy - Reaction center complex- organized association of proteins holding a special pair of chlorophyll a molecule. o Contains a molecule capable of accepting electrons and becoming reduced (primary electron acceptor)- pair of chlorophyll a molecules are special because of their environment- able to use energy from light to boost electron to higher energy but also to transfer it to the primary electron acceptor. - Light harvesting complex- various pigment molecules (chlorophyll a, chlorophyll b, carotenoids) bound to proteins o Number and variety of pigment molecules helps photosystem harvest light over bigger surface area and act as antenna for reaction center complex o When pigment molecule absorbs light, the photon is passed along from molecule to molecule until it is passed into reaction center complex. - Transfer of photon from reaction center chlorophyll a pair to primary electron acceptor is first step of light reactions. Excited then primary electron acceptor catches it which happens in structured environment of chloroplast (redox reaction). - Thylakoid is populated by two types of photosystems that cooperate in the light reactions of photosynthesis- photosystem II and photosystem I. o Reaction center of chlorophyll a in PSII is known as P680 because it is best at absorbing light with wavelength of 680. o Chlorophyll at reaction center complex of PSI is known as P700 because it is best at absorbing light with wavelength of 700. o They have different proteins associations which effects electron distribution which accounts for slight differences in light absorbing properties Linear Electron Flow - Light drives synthesis of ATP and NADPH by energizing the two photosystems, but a flow of electrons through the photosystems and other components in thylakoid is necessary. - Steps- - 1. Photon hits pigment in PSII and an e- is boosted to higher energy level and as this falls another e- in a nearby pigment is excited- repeats until it reaches P680, and an electron pair of chlorophylls is excited - 2. E- is transferred from excited P680, which is now known as P680 + (strongest oxidizing agent), to primary electron acceptor - 3. Enzyme catalyzes split of water into H+, 2 e-, and O atom. The e- are supplied one by one to P680, H+ go into thylakoid lumen, and O combines with another O to make O 2 - 4. Each photoexcited electron is passed from primary electron acceptor of PSII to PSI via ETC made up of proteins, which is exergonic. - 5. ETC provides energy for synthesis of ATP, and H+ are pumped into thylakoid lumen, helping with proton gradients that is used in chemiosmosis. - 6. Light energy has been transferred via light harvesting complex pigments to the PSI reaction center complex, exciting an electron of the P700 pair of chlorophyll a, which was then transferred to PSI’s primary electron acceptor, making it P700+ (a hole/missing something), allowing it to act as an electron acceptor, accepting an electron that reaches the bottom of the ETC (gets more electronegative as it gets toward bottom and source for electrons is water) from PSII. - 7. Redox reactions occur when pass from primary acceptor down second ETC through protein Fd (no proton gradient, so no ATP) - 8. NADP+ reductase (enzyme) catalyzes transfer from Fd to NADP+ and made into NADPH by two electrons. NADPH is higher energy than water and e- more readily available for Calvin cycle. Removes H+ from stroma Cyclic Electron Flow - only uses PSII in certain cases o electrons cycle back from Fd to cytochrome complex (which makes ATP via chemiosmosis) and then to P700 no production of NADPH, no release of O , but it generates 2 ATP Comparison of chemiosmosis in mitochondria and chloroplasts is explained in picture Summary of light reaction- - electron flow pushes electron from water (low state of energy) to NADPH (stored as high state of energy) and this current drives production of ATP. O i2 byproduct Calvin Cycle - uses chemical energy of ATP and NADPH to reduce CO to sugar 2 - anabolic- builds carbohydrates from smaller and consumes energy - Carbon enters in form of CO and2leaves as sugar. Spends ATP as energy source and consumes NADPH as reducing power for adding high energy electrons to make sugar. - Carbohydrate produced directly is glyceraldehyde 3-phoshphate G3P- for synthesis of one, Calvin cycle must happen three times (fixing 3 CO 2olecules) - Phase 1- Carbon Fixation- gets each CO molecules2by attaching it to five carbon sugar RuBP, which is catalyzed by rubisco (most abundant protein), which makes a very unstable 6C which then splits in half forming two 3-phosphoglycerate (for each CO ) 2 - Phase 2- Reduction- each 3-phosphoglycerate gets phosphate group from ATP, making it 1, 3-bisphoglycerate; then a pair of electrons from NADPH reduces that and makes it lose a phosphate group, which then becomes G3P (same sugar from glycolysis when glucose is split). So, 6 G3P are formed for all three CO 2 - Phase 3- regeneration of the CO acceptor2(RuBP)- the carbon skeleton of five molecules of G3P are rearranged into three molecules of RuBP by spending three more molecules of ATP and RuBP is now prepared for more CO 2 - Net synthesis of one G3P- consumes 9 molecules of ATP and 6 NADPH, but light reaction regenerates ATP and NADPH. G3P made from Calvin cycle is string material for for metabolic pathways that synthesize other organic compounds. Importance of Photosynthesis - from photons to food - light reaction capture solar energy and make ATP and transfer electrons from water to NADP+ to form NADPH. The Calvin cycles uses the ATP and NADPH to produce sugar and carbon dioxide. Energy that enters chloroplasts as sunlight becomes sotred as chemical energy in organic compounds. - Sugar gives entire plant chemical energy and carbon skeleton for production of organic molecules in cell- half of organic material is consumed as fuel for cellular respiration - Responsible for oxygen in atmosphere - Makes billions and billions of carbohydrates Chapter 16- Molecular Basis of Inheritance The Search for Genetic Material (DNA) - Early in 20 century, identifying molecules of inheritance was a major challenge - Morgan showed that gene exists as part of chromosomes (made of DNA and proteins), so they were tested on for genetic material o Before scientists were testing proteins, but it came out as different results, so they knew they were wrong, so when Morgan and Mendel figured it out, it was revolutionary o Worked on bacteria to find out this information Evidence DNA Can Transform Bacteria - 1928- attempt to make vaccine against pneumonia, Griffith was studying the bacteria that causes pneumonia in mammals o Had two strains of bacteria- one pathogenic (causes disease) and other non-pathogenic (harmless) Surprising- when he killed the pathogenic bacteria of nonpathogenic strain, some of the living cells became pathogenic, so that means that pathogenicity was inherited by decedents of the bacteria. Clearly some chemical of the dead pathogenic cell caused the heritable change Transformation- change in genotype and phenotype because of assimilation of external DNA by a cell. - Avery was now inspired o Focused on DNA, RNA (other nucleic acid in cell), and proteins o Broke open the heat killed pathogenic bacteria and took cellular contents o Took DNA, RNA, and protein and inactivated one of them and tested its ability to transform live nonpathogenic bacteria Only when DNA was active, the transformation occurred Hence, in 1944, it was announced that DNA is the transforming agent, but many scientists still doubted it Evidence that Viral DNA Can Program Cells - More evidence that DNA was the genetic material came from studies that infect bacteria= bacteriophages (aka phage) - Virus- is just a little more than DNA with a protective coat around it, mostly a protein o to produce viruses, the virus must infect a cell and take over the metabolic machinery - 1952- Hershey and Chase showed that DNA is genetic material of phage (T2) which infects e coli, so they experimented on e coli o At that time, they knew that T2 was made up mostly DNA and protein and that T2 can infect e coli and make it release many more T2 when it breaks, but needed to find out if it was protein or DNA Used radioactive isotope of sulfur to tag to protein in one and DNA in another, and they found out that the phage DNA entered the host cell but phage protein did not. With the DNA, it let out many others with the virus. o Concluded that DNA is the molecule to carrying genetic information that makes cell produce new viral DNA and protein. – clear evidence that it is DNA and not protein Additional Evidence that DNA is the Genetic Material - Chargaf- already known that DNA is polymer of nucleotides, each having a nitrogenous base (A, T, G, or C), pentose sugar of deoxyribose, and a phosphate group Rules that were explain o Base composition varies from one species to another when double helix came o Number of A=T and number of G=C along Building Structural Model of DNA - Now- how could structure fit role of inheritance - 1950s- arrangement of covalent bonds in nucleic ace polymer was known - 1953- Watson and Crick- used X-ray crystallography and studied protein structure. Watson saw an Xray defraction picture of DNA produced by Franklin, and a picture produced by xrays that were deflected as they passed through aligned fibers of DNA, which were both helical because of defractions, which just confirmed his beliefs that DNA was helical- made up of two strands= double helix. o Started to make models of double helix which would conform to xray measurements and the known chemistry of DNA (including Chargaff and with Franklin’s-female- information saying that sugar phosphate backbones were on outside of DNA) Hydrophobic parts were in interior and nitrogenous Like bases on interior twisted Negatives charges weren’t near each other ladder Sugar phosphates were antiparallel- subunits run in opp direction A-T, and G-C (need a purine and pyrimidine to be right size) DNA Replication and Repair Basic Principle- Base Pairing to a Template Strand - Watson and Crick hypothesis- two strands that are complementary to each other o Before it duplicates, hydrogen bonds are broken, and two chains unwind and separate, and then each acts as template for new chains, so it would be 2 exact same strands Nucleotides just match up to complementary strand Each daughter molecule will have one old parental strand and one new strand- semiconservative model compared to conservative model where both strands just come back together after process and compared to dispersive model where all four strands have some new and some old Meselson and Stahl confirmed the semiconservative model DNA Replication - E coli has one chromosome with 4.6 million nucleotide pairs, and can replicate this DNA in less than an hour, but we have 46 DNA molecules in each cell in nucleus with long double helix in chromosome, which in all has 6 billion nucleotide pairs. - Proteins and enzymes help it along - Step 1- begins at origins of replication (short stretches of DNA with a specific sequence of nucleotides), and then proteins attach to the DNA there and separate the strands making a replication bubble. Replication proceeds in both directions until entire molecule is copied. o Bacterial chromosomes only have one origin, while eukaryotic have many- multiple bubbles form with each other and fuse together, speeding up process Both- replication proceeds in both directions from each origin o At end of replication bubble is a replication fork- Y shaped region where parental strands of DNA are unwound (the untwisting, twists the twisted part even more- pulls it tighter) Helicases- enzymes that untwist the double helix at replication forks, making them available to be a template strand Single strand binding proteins- bind unpaired DNA strands, keeping them from repairing with each other Topoisomerase- helps relieve tightening the twisted strands by breaking, swiveling, and rejoining DNA strands. o Now a template, but enzymes that synthesize DNA cannot start synthesis of polynucleotide, rather they can only add nucleotides to the end of existing chain that is base-paired with template. Initial nucleotide chain that is produced during DNA synthesis is rally RNA= primer and synthesized by primase, starts complementary RNA strand from single RNA nucleotide, adding RNA nucleotides one at a time, using the DNA template, which is then base paired to template and will start from 3’ end of RNA primer. - Step 2- Enzyme DNA polymerase- catalyze synthesis of new DNA by adding nucleotides to preexisting chain- bacteria have 2 while eukaryotes have 11 o Require primer and DNA template strand, and add DNA nucleotides to RNA primer and then continue to DNA strand o Each nucleotide added becomes a nucleoside triphosphate (sugar and base with three phosphate), just like ATP but with deoxyribose instead of ribose, so dATP that supplies adenine nucleotide to DNA Chemically reactive because triphosphate tail has unstable cluster of neg charge As each monomer joins, two phosphate groups are lost as pyrophosphate, which is the hydrolyzed with two molecules of inorganic phosphate and is a coupled exergonic reaction that helps drive the polymerization reaction o 2 new strands are antiparallel to the template too Primases can only add to the free 3’ end of primer or growing DNA strand, not 5’ end. Therefore, new DNA can only elongate in 5’ to 3’ direction Leading Strand- DNA strand made by adding nucleotides (the complementary strand) Lagging Strand- the other DNA strand is the mandatory 5’ to 3’ direction, so DNA polymerase must work along the template strand in direction away from the replication fork because can only add in 5’ to 3’, but has to be antiparallel It adds nucleotides discontinuously, in segments= Okazaki fragments 1. Primase joins RNA nucleotides into primer DNA polymerase III adds DNA nucleotides to primer of fragment 1 After it touches the next primer, polymerase detaches Fragment 2 is primed, and DNA polymerase adds DNA nucleotides, detaching when it reaches the fragment 1 primer DNA polymerase I replaces RNA with DNA, adding 3’ end of fragment 2 DNA ligase forms a bond between the newest DNA and the DNA fragment 1- joins final nucleotide of replacement DNA to first DNA nucleotide of adjacent Okazaki fragment The lagging strand in this region is now complete o DNA polymerases are represented as trains moving down a track, which is wrong because: Many proteins interactions facilitate form a large DNA complex Primase acts as a brake at the fork when other proteins interact with it and slows down the process and coordinates the placement of primers and rate of replication on both strands DNA replication complex may not move, rather the DNA moves through the complex- lagging strand loops back around through the complex DNA Proofreading and Repairing - DNA polymerases proofread each nucleotide with template- if incorrect, then it will remove the nucleotide and resume to synthesis - Mismatch Pair- other enzymes remove and replace incorrectly matched enzymes o This pair can cause cancer - Incorrectly or altered pairs- can damage DNA if not repaired, bad pairs happen often, but usually fixed before permanent; if they aren’t, it is called a mutation o Many enzymes are present to try to fix them o Most use mechanism that takes advantage of the base-paired structure of the DNA Nuclease- the DNA cutting enzyme that takes out incorrect pair, then the resulting gap is filled with other nucleotides via DNA polymerase or DNA ligase, using undamaged strand as template- this whole thing is called nucleotide excision repair Replicating the Ends of DNA Molecules - Because DNA polymerase can only add to the 3’ or a preexisting template, there is no way that it can complete the full replication of a daughter DNA strand. Even if it is started with RNA primer, the primer is removed, and then it cannot be added before because of the 5’ end; therefore, repeated rounds of replication produce short DNA molecules with uneven/staggered ends. Therefore, eukaryotic chromosomal DNA molecules have special nucleotide sequences called telomeres at end. o Telomeres don’t have genes, but repeat certain nucleotide sequence many times, which then protects the organisms gene o Proteins associated with telomeres prevent staggered ends of daughter cells from activating, which prevents damage o They become shorter during every round of replication o Telomerase catalyzes the lengthening of telomeres so genes are not lost and chromosomes are not shortened o Shortening of telomeres can protect organisms from cancer by limiting divisions that somatic cells can undergo Cells from large tumors have short telomeres, since it underwent cell divisions, and further shortenings would lead to self destruction of tumor cells, but telomerase would allow these cancer cells to persist because it wouldn’t make the telomerase shorter, so telomerase is imp in cancer cells - Prokaryotes have circular chromosome so shortening does not occur Chromosome - Chromosome- consists of DNA molecule packed together with proteins o They are coiled- densely packed in there, so it is really a lot bigger than cell if it was stretched out o In bacteria it is in the nucleoid- which is a not bounded membrane o In eukaryotic cells- single DNA double helci averages about 1.5 x10 nucleotide pairs and would be 4 cm if stretched out In Eukaryotic cells, DNA is combined with protein, which together make make up a complex called chromatin, which fits into the nucleus. Chromatin Packing in Eukaryotic Chromosomes - DNA and the phosphate groups have neg charge, which is on the outside of each strand - Histones- small proteins that are responsible for first level of DNA packing in chromatin o Positively charged so bond to DNA o DNA wraps around, so it structures and orders DNA into nucleosomes - Nucleosome- basic unit of DNA packing o each individual wrapping, the individual bead on a string o DNA wound twice around a protein core composed of two molecules of each of the four main histones, and the histone tail (N terminus) is facing outward - Interactions between histone tails and linker DNA and nucleosomes on either side o 5 histone is involved o the interactions cause the fiber to coil, forming a chromatin fiber with 30nm in thickness - The fiber then forms a loop called looped domain, attached to a chromosome scaffold made of protein (together=300nm fiber) - In mitotic chromosome- looped domain coil and fold, which compacts all chromatin, which then makes the classic chromosome picture of 1400nm (each chromatid 700nm) Chapter 17 From Gene to Protein Overview of Flow of Genetic Information - DNA inherited by organism leads to specific traits by dictating synthesis of proteins and of RNA molecules involved in protein synthesis - Gene expression- process by which DNA directs synthesis of proteins o two stages- transcription and translation Garrod- first to suggest that genes dictate phenotype through enzymes that catalyze specific reactions, disease can be caused by not being able to create a certain enzyme - genes dictate production of enzyme Many eukaryotic genes can code for a set of closely related polypeptides via alternative splicing. Few genes code for RNA molecules that are imp to cell but not translated into protein. Basic Principles of Transcription and Translation - Gene does not build protein directly, just give instructions - Nucleic acid RNA (ribose and U base and single strand) is bridge between DNA (deoxyribose and T base and double strand) and protein - Both nucleic acids and proteins are polymers that convey information o DNA/RNA- monomers are 4 types of nucleotides that differ in bases and genes are many nucleotides long o Proteins- monomers are amino acids o Both have different languages but convey information, so need transcription and translation to convert from DNA to protein Occurs in both Transcription- synthesis of RNA using information from DNA, eukaryotes and prokaryotes, just in rewritten in RNA form prokaryotes they - DNA template serves as template for making the happen at same complementary RNA strand, time because not o For protein coding gene- it is mRNA that is synthesized organelles but in eukaryotes it is at since it carries genetic information from DNA to protein different times- synthesizing machinery transcription in o Eukaryotes- it makes pre-RNA or primary transcript nucleus and and then other processes occur to make it mRNA translation in cytoplasm Translation- synthesis of polypeptide using information from mRNA - Change in language- nucleotide sequence to amino acid - Occurs in ribosomes Central Dogma- DNA to RNA to protein Genetic Code - only four nucleotide bases but 20 amino acids, so how can it be that DNA makes specific protein? - multiple nucleotides correspond to one amino acid. - Triplets of nucleotide bases are the smallest number of units that can code for amino acid, allows there to be 64 (4 ), which is more than enough combinations= triplet code- series of words in a gene is transcribed into complementary series of three nucleotide words in mRNA, then translated into amino acid. o mRNA nucleotide triplets= codon and written in 5’ to 3’ o more than one codon can indicate one amino acid o need to make sure it is read correctly because if one base is missing, everything is thrown off - In transcription- only one strand of DNA is being transcribed= template strand, which provides pattern for sequence on nucleotides in an RNA transcript - mRNA is complementary, not identical, to template- base pairing, synthesized antiparallel o U is in RNA instead of T in DNA as a base and ribose is in RNA instead of deoxyribose in DNA as a sugar o 3’-ACC-5’ synthesizes 5’-UGG-3’ - During translation- codons are decoded/translated into sequence of amino acids o Codons are read in 5’ to 3’ along mRNA o If there are 300 nucleotides, there will be 100 amino acids in polypeptide chain o 3 codons- do not designate amino acids- stop signals to end translation- UAA, UAG, UGA o start codon- initiations signal- mRNA codon AUG, to begin translating Transcription DNA directs synthesis of RNA mRNA (carrier of information from DNA to cell protein synthesizing machine) is transcribed from template gene - RNA polymerase- enzyme separates two strands of DNA and joins RNA nucleotides complementary to DNA template strand o Can only assemble in 5’ to 3’ direction o Able to start from scratch, no primer necessary - Promoter- DNA sequence where RNA polymerase attaches and starts transcription - Terminator- signal that ends transcription (downstream from promoter) - Transcription unit- the stretch of DNA that is transcribed into an RNA sequence - Prokaryotes have one type of RNA polymerase and eukaryotes have three types o mRNA synthesis is RNA polymerase II - Initiation- promoter of gene includes- start point- nucleotide where RNA synthesis actually happens- and extends several dozen or more nucleotide pairs upstream from start point. RNA polymerase binds to promoter and determines where transcription starts and which strand is used as template o Bacteria- RNA polymerase binds to promoter itself o Eukaryotes- collection of proteins (transcription factors) mediate binding of RNA polymerase and initiation of transcription- once transcription factors are attached to promoter, then RNA polymerase II binds to it. Whole complex= transcription initiation complex. TATA Box- crucial promoter DNA sequence Protein-protein interactions are crucial in controlling eukaryotic transcription- ex- interaction between eukaryotic RNA polymerase II and transcription factors - Elongation- RNA polymerase moves along DNA and untwists the double helix, it adds nucleotides to 3’ end of growing RNA molecule, synthesizing RNA and peels away from DNA template and DNA double helix reforms. o More than one polymerase can transcribe a gene, which increase amount of mRNA made, which helps make more protein - Termination- bacteria- transcription goes through terminator sequence in DNA, and the transcribed terminator (the RNA sequence of termination) is the signal and causes polymerase to detach from DNA and release the transcript o Eukaryotes- RNA polymerase II transcribes sequence of DNA called polyadenylation signal (AAUAAA) in pre-mRNA and then at a point 10-35 nucleotides downstream from it, proteins associated with growing RNA transcript cut it free from polymerase, releasing the pre-mRNA which undergoes RNA processing Eukaryotic Cells Modify RNA after Transcription - With pre-mRNA om nucleus, before cytoplasm and it is called RNA processing - Alterations of mRNA ends- 5’ end gets a 5’ cap, modified form of a guanine (G) nucleotide added on the 5’ end after transcription of first 20-40 nucleotides. o 3’ end- enzyme adds 50-250 A nucleotides forming poly-A tail o both of them- facilitate export of ready mRNA, protect, help ribosomes attach to 5’ end of mRNA once it reaches cytoplasm o UTRs- ends of the 5’ and 3’ ends, but not translated into proteins, but helps with ribosome binding - Split Genes and RNA Splicing-RNA splicing- removal of large portions of RNA that is initially synthesized, which are not continuous with each other. Cut out introns but keep exons, part that is expressed in protein, except for UTR- exons exit nucleus o snRNPS (in nucleus and contain RNA and protein molecules) recognize splice sites (has RNA in there called snRNA and help catalyze) can join with other proteins to create a spliceosome- almost the size of ribosome, interacts with cites along the intron and releases the intron, and joins the two exons together, creating mRNA o Ribozymes- RNA molecules that function as enzymes, some can splice introns out by themselves 3 reasons why RNA molecules can function as enzymes RNA is single stranded (and can base pair with others) gives it a particular shape Has similar functional groups to enzyme Can hydrogen bond with other nucleic acid molecules adds specificity to its catalytic activity - Alternative RNA splicing- many genes create two of more polypeptide, depending on which sections are treated as exons during RNA processing o Number of proteins produced can be much more than number of genes because of this - Domain- distinct structure and parts of proteins- exons can code for it o One domain might include active site, while another might allow enzyme to bind to cellular membrane - Exon shuffling- exons crossing over to create different protein- mixing with one another Translation - RNA directed synthesis of polypeptide (protein) - Message is the series of codons along an mRNA, and the translator is tRNA (80 nucleotides long)- transfer amino acids from cytoplasmic pool of amino acids to growing polypeptide in ribosome o Ribosomes (made of RNA and protein) adds amino acids brought to it by tRNA to growing end of polypeptide chain o Molecules of tRNA are not all identical and each part of tRNA translates a particular mRNA codon into an amino acid o tRNA arrives are ribosome with amino acid on one side and anticodon (base pairs with a complementary codon on mRNA) on other o codon by codon message is translated as tRNAs deposit amino acids in order prescribed and ribosome joins amino acid into chain o tRNA is translator- read nucleic acid word and interpret it as protein word o tRNA is transcribed from DNA template- eukaryotes- made in nucleus and then goes to cytoplasm where translation occurs- picking up amino acids in cytosol and then adding it to polypeptide chain at ribosome and then leaving ribosomes ready to pick up another amino acid o stretches of nucleotide bases that can hydrogen bond to each other and can fold back onto itself because it is single strand and for a 3D structure (cloverleaf) and then from there twists and fold into L-shaped- at the bottom has an anticodon (base pairs to mRNA codon) and then the other side (5’ end) is the attachment site for amino acid. Anticodons are written in 3’ to 5’ to align with the 5’ to 3’ codons 3’AAG5’ anticodon pairs with mRNA codon 5’UUC3’ o two instances of molecular translation- 1. tRNA that binds to mRNA codon specifies the amino acid that it has to bring to ribosome aminoacyl-tRNA synthecase- atches up the correct tRNA and amino acid active site fits only a specific amino acid sequence and tRNA 20 different ones- ones for each amino acid- can bind to tRNAs that codes for amino acid catalyzes covalent bond of tRNA and amino acid by hydrolysis of ATP, so now it is a charged tRNA, which is then released from enzyme and available to deliver amino acid to polypeptide chain of ribosome pairing of tRNA anticodon with appropriate mRNA codon some tRNAs can bind to more than one specific codon- U can pair with A or G because of the phenomenon wobble- flexible base pairing at specific codon position (U at 5’ end of anticodon can pair with either A or G at 3’ of codon)- explains synonymous codons given for amino acids most often differ in third nucleotide base (anticodon 3’UCU5’ can base pair with mRNA codon 5’ AGA 3’ or 5’AGG3’) - Ribosomes- facilitate specific coupling of tRNA anticodons with mRNA codons during protein synthesis. Both subunits are made up of proteins and rRNA o Eukaryotes- subunits are made in nucleolus o Ribosomal RNA genes are transcribed and RNA is processed and assembled with proteins from cytoplasm and the resulting ribosomal subunits are then exported via nuclear pores to cytoplasm Subunits only function when they attach to mRNA molecule (bacteria and eukaryotes)- rRNA is most abundant RNA Eukaryotes’ ribosomes are slightly larger than prokaryotes- differences are significant though small- antibiotic can inactive bacterial ribosome but not inhibiting eukaryotic ribosomes to make proteins. o Structure reflects function of bringing mRNA together with tRNAs carrying amino acids Has binding site for mRNA and thre binding sites for tRNA P site- hold tRNA carrying growing polypeptide chain A site- holds tRNA carrying next amino acid to be added to chain E site- tRNAs leave ribosomes through this Holds tRNA and mRNA close to each other and positions new amino acid for addition of carboxyl end of growing polypeptide, and then catalyzes formation of peptide bond. Polypeptides goes through exit tunnel in large subunit - Ribosome Association and Initiation of Translation- brings mRNA (tRNA bearing first amino acid of polypeptide) and two subunits of ribosome together o Ribosomal subunit binds to mRNA and specific initiator tRNA Bacteria- can happen in either order- binds to mRNA upstream of AUG Eukaryotes- small subunit with initiator tRNA already bound binds to 5’ cap of mRNA and moves/scans downstream along mRNA until it reaches start codon and then the initiator hydrogen bonds to AUG start codon, which signals translation, & establishes codon reading frame for mRNA o After mRNA, initiator tRNA, and small subunit unite, attachment of large ribosomal subunit occurs= completion of translation initiation complex Proteins help bring everything together Cells spends energy obtained by hydrolysis of GTP to form the complex tRNA now sits in P site and A site is ready for next tRNA polypeptide is always synthesized from methionine amino acid end (N terminus) to final amino acid end (carboxyl terminus) - Elongation of Polypeptide Chain o Amino acids are added one by one to previous amino acid at C terminus o Needs proteins called elongation factors to add o 3 steps 1. Codon recognition- anticodon of tRNA base pairs with mRNA codon in A site. Hydrolysis of GTP increases accuracy and efficiency of this step. 2. Peptide bond formation- rRNA molecule of large subunit catalyzes formation of peptide bond between amino group of amino acid in A site and C end of growing polypeptide in P site. This removes polypeptide from tRNA in P site and attaches it to amino acid on the tRNA in A site. 3. Translocation- ribosome translocates the tRNA in A site to P site and at the same time, the empty tRNA in P site is moved to E site to be released. mRNA moves along with its bound tRNAs, bringing next codon to be translated into A site o GTP hydrolysis is used in step 1 (codon recognition) and 3 (translocation). o Moves 5’ to 3’- just like mRNA - Termination of Translation- elongation happens until stop codon of mRNA reaches A site o UAG,UAA, and UGA are stop codons, don’t translate into protein o Release factor- protein shaped like tRNA- binds directly to stop codon in A site and calls for addition of water instead of amino acid to polypeptide chain, which breaks bond (hydrolyzes) of completed peptide and tRNA in P site, releasing polypeptide through exit tunnel in large subunit o Remainder of translation then comes apart in multistep process, aided by protein factors and requires hydrolysis of two more GTP molecules. - Single mRNA is used to make many polypeptides at the same time- once ribosome is far enough passed start codon, another can attach to mRNA and translate= polyribosome - Completing and Targeting the Functional Protein- modifications & finishing polypeptides o Protein Folding and Post Translational Modification- Synthesis- polypeptide chain begins to coil and fold spontaneously because of amino acid sequence (primary sequence), forming protein with specific 3D shape (secondary and tertiary), chaperone helps it fold correctly o Post-translational modifications- other substances can be added to amino acids Enzymes can remove amino acids from leading end of chain Enzymes can cleave o Free ribosomes are in cytosol and synthesize proteins that usually stay there o Bound ribosomes are attached to inner side of ER, make proteins of endomembrane system and proteins secreted from cell o Polypeptide synthesis always begins in cytosol and starts to translate mRNA and there the process is completed unless the growing polypeptide itself cues ribosome to attach to ER. Ones for secretion are marked by signal peptide- targets protein to ER where it can finish translation Recognized as it emerges from signal recognition particle (SRP) which is a ribosome by a protein RNA complex with a membrane pore and signal cleaving enzyme- once SRP leaves, protein synthesis continues and is still attached to translocation complex The signal cleaving enzyme cuts off the signal peptide and the rest of the completed polypeptide leaves ribosome and folds into final form Escorts ribosome to receptor protein in ER Chapter 18- Regulation of Gene Expression - Everyone must change pattern of gene expression in response to environment - Each cell contains same genome but expresses different genes - Usually regulated during transcription, but still need control at different stages Bacteria Often Respond to Environmental Change by Regulating Transcription - Cells don’t want to work so much, so natural selection favors bacteria that express only the genes whose products are needed by cell (save energy) - Ex- E coli lives in human, but needs amino acid tryptophan to survive, so cell responds by activating metabolic pathway to make tryptophan, but once the human eat something with tryptophan, the metabolic pathway will stop. (change based on enviro) - Two levels of metabolic control- o Cells can adjust activity of enzymes present- which is fast Feedback inhibition- end product said that there is too much, so it stops the anabolic pathway (creation) of that product from the first enzyme- makes cell adapt- make quick changes based on cells’ needs o Cells can adjust production of certain enzymes- regulate expression of genes coding for the enzyme If too much of something, the cell will repress expression of the genes encoding for the enzyme that creates the product Occurs in transcription- synthesis of mRNA coding for enzyme Operons - The cell can translate one mRNA into five peparate polypeptides, which make up the different enzymes because of the start and stop codons that signal different polypeptides. - A transcription unit groups genes of related functions together, and therefore only have one “on-off” switch that can control the whole cluster - The switch is the operator (DNA segment)- o Positioned with promoter or between enzyme coding genes o Controls access of RNA-polymerase to genes (allows transcription) - Promoter, operator, and genes they control= operon o Trp operon is one of many in E coli genome - Operator is operon’s switch for controlling transcription- o By itself, trp operon is turned on- RNA polymerase can bind to promoter and transcribe genes of operon o Can be turned off by a protein called trp repressor- binds to operator and blocks attachment of RNA polymerase to promoter (can’t transcribe) Specific for operator of particular operon Trp repressor is protein product of regulatory gene called trpR, located near trp operon and has own promoter. Expressed continuously at a low rate, but not switched off permanently because o Binding of repressors to operators is reversible- operators waver between a state of without repressor bound and a state of being repressor bound, and the time of each state depends on the amount of repressors there are around. Also regulatory proteins are allosteric, with an inactive shape and an active shape. Trp repressor is made in an inactive form with little affinity for trp operator- only if trp binds to trp repressor at an allosteric site, then the repressor protein changes to active site and attaches to the operator, turning the operon off. o Corepressor- a small molecule that cooperates with a repressor protein to switch operon off. Trp functions as one- as trp accumulates, trp molecules associate with trp repressor molecules, which then bind to trp operator and shuts the trp pathway enzymes off. If trp levels drop, then transcription of operon’s genes resume. Trp repressor is inactive by itself and need tryptophan as a corepressor in order to bind to operator Repressible and Inducible Operons- - Repressible Operon- inhibits transcription when small molecule binds allosterically to regulatory protein (trp operon) - Inducible Operon- usually off but can be stimulated when specific small molecule interacts with a regulatory protein (lac operon) o Lactose is available to E coli in human colon if human drinks milk Metabolism begins with hydrolysis of the disaccharide into glucose and galactose, which is catalyzed by an enzyme, and only a few molecules of that enzyme are present in an E coli cell growing in absence of lactose. But if lactose is added, then the number of that enzyme increases majorly. Active by itself, and binding to operator and switching the lac operon off This is an example of inducer (inactivates repressor), and for lac it is allolactose (inducible enzymes) In absence of lactose (and allolactose), lac repressor is in active configuration, and gene of lac operon are silenced. If lactose is there, then allolactose binds to lac repressor and changes conformation, nullifying repressor’s ability to attach to operator. Without bound repressor, the lac operon is transcribed into mRNA for the lactose- utilizing enzyme. Repressible enzymes function in anabolic pathways- suspend end product, and use energy for other uses Inducible enzymes- catabolic pathways, producing enzymes when nutrient is available Both negative control of genes because it is turned off by active form of repressor protein Positive Gene Regulation - E coli prefers glucose over any other sugar. When glucose is not present, it will use lactose as energy source - It can tell that glucose is not there and relays it to gene by- o Allosteric protein interacts with cAMP, small protein that accumulates when glucose is scarce. o The regulatory protein CAP (catabolite activator protein) is an activator, which is a protein that binds to DNA and stimulates transcription of a gene. When cAMP binds, CAP assumes active shape and can attach to specific site upstream end of lac promoter This increases the affinity of RNA polymerase for promoter, which is low when no repressor is bound to operator. Attachment of CAP, directly stimulates gene expression- binding of RNA polymerase to promoter which increase transcription o If amount of glucose increases, cAMP concentration falls; without cAMP, CAP detaches from operon, and is inactive, so RNA polymerase attaches less efficiently to promoter, and transcription of lac operon proceeds at low level even in presence of lactose. Lac operon is under negative control by lac repressor and positive control by CAP State of lac repressor (w/ or w/o allolactose) determines if transcription will occur State of CAP (w/ or w/o cAMP) control rate of transcription if operon is repressor free o CAP helps regulate other operons that encode enzymes used in catabolic pathways o When glucose is plenty, and CAP is inactive, synthesis of enzymes that catabolize compounds other than glucose generally slows down Compounds present in the cell at the moment determine which operons are switched on Eukaryotic Gene Expression is Regulated at Many Stages Differential Gene Expression - Human cells only show 20% of their genes- all cells have same genome, except show it differently and different ones are expressed, allowing the different functions - Diferential gene expression- expression of different genes by cells with same genome - Transcription factors of cell must locate right genes at right time o Regulation of Chromatin Structure DNA of eukaryotic cells is packaged proteins= chromatin It helps regulate gene expression- location of gene’s promoter relative to nucleotsomes and to site where DNA attaches to the chromosome scaffold can effect if gene is transcribed and chemical modifications to histone proteins and to DNA of chromatin can influence the chromatin structure and gene expression Histone Modification- histone= protein in which DNA is wrapped in nucleosomes N terminus faces outwards of the nucleosome and are accessible to various modifying enzymes that catalyze addition/removal of specific chemical groups o Histone Acetylation- acetyl groups (COCH ) 3 are attached to lysine in histone tails; when they are acetylated, the lysine positive charge are neutralized and histone tails no longer bind to neighboring nucleosome, which promotes folding of chromatin into more compact structure, so transcription proteins have easier access to genes in acetylated region Associated with components of transcription factors that bind to promoter- may promote initiation of transcription o Methyl can promote condensation of chromatin, while addition of phosphate group near methylated group can have opposite effect o DNA Methylation- different set of enzymes can methylate certain bases in DNA itself (don’t change DNA sequence) long stretches of inactive DNA are usually more methylated than active DNA, but on a smaller scale, single genes are more methylated in cells that they aren’t expressed now- removal of methyl groups will turn on some genes essential for long term inactivation during normal cell differentiation in embryo genetic imprinting- methylation permanently regulates expression of allele of genes methylation patterns are passed on to next generation o Epigenetic Inheritance- inheritance of genes not directly involving nucleotide sequence Modifications in chromatin can be reversed (can silence a specific gene) DNA methylation and histone deacetylation can repress transcription Trying to figure out why with identical twins, one has something but other does not, and seem in some cancers Enzymes that modify chromatin structure are integral for regulating eukaryotic transcription Regulation of Transcription Initiation - Chromatin modifying enzymes provide initial control of gene expression by making a region of DNA more or less able to bind to transcription machinery - Once chromatin is modified for expression, transcription initiation regulation can occur Typical Eukaryotic Gene - Transcription initiation complex comes on promoter sequence upstream end of gene - RNA polymerase II (one of the proteins) transcribes the gene, making pre-mRNA - 5’ Cap is added and poly-A tail, splicing out introns= mature mRNA - control elements- segments of noncoding DNA that serve as binding sites for transcription factors (regulate transcription) - Roles of Transcription Factors o RNA polymerase needs it to start initiation process o A few independently bind a DNA sequence (TATA box with promoter), usually bind proteins, which is crucial- allows RNA polymerase to go o Some are general and some are specific o Proximal control elements- located close to promoter o Distal control elements= enhancers- many nucleotides away (upstream or downstream) Gene can have many, activated at diff times and locations, but specific for gene o Rate of transcription is increased/decreased by binding of transcription factors (activators or repressors) to control elements of enhancers o Two common structural elements in activator proteins- a DNA binding domain and one or more activation domains (which bind other regulatory proteins or components of transcription Same gene is machinery that result in transcription of gene) expressed in o Binding activators to distal enhancers can effect transcription- certain tissue but Activator proteins bind to distal control elements grouped not others as enhancer in DNA, which has three binding sites, each The DNA of both liver and called distal control elements lens cells contain the DNA bending protein brings bound activators closer to genes for both the protein albumin and the protein promoter. General transcription factors, mediator proteins, crystallin and RNA polymerase II are close However, the albumin gene Activators bind t certain mediator proteins and general is expressed only in liver transcription factors, helping them form an active while the crystallin gene is expressed only in the eye transcription initiation complex on promoter In other words, the proteinription factors that act as repressors inhibit gene albumin is synthesized onlyssion by binding directly to control element DNA in the liver (although(enhancers sometimes), blocking activator binding, or turning off then transported to thtranscription. blood) while the protein crystallin is synthesized in Some effect indirectly- affect chromatin structure- acetylate histones near promoters of genes, which promotes transcription
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