Study Guide for Biochem Exam 2
Study Guide for Biochem Exam 2 87222 - BCHM 3050 - 002
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This 31 page Study Guide was uploaded by America Seach on Friday February 19, 2016. The Study Guide belongs to 87222 - BCHM 3050 - 002 at Clemson University taught by Srikripa Chandrasekaran in Spring 2016. Since its upload, it has received 247 views. For similar materials see Essential Elements of Biochemistry in Biology at Clemson University.
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Date Created: 02/19/16
Biochem Lecture 7: - Nucleic Acid: Linear polymers of nucleotides that function in the storage and expression of genetic information, and its transfer from one generation to the next. - Two main types: Ribonucleic Acid (in RNA) and De-oxyribonucleic Acid (in DNA) - Molecular components of nucleotides (three components) 1. Nitrogenous base a. 1’ is always connected to the nitrogenous base 2. Pentose sugar 3. Phosphate a. 5’ is always connected to the phosphate - 2’ is always the one that distinguishes whether it is part of RNA (has an OH) or DNA (only has H) - Nitrogenous Base families: - o Pyrimidines: six-membered heterocyclic rings of C and N o Purines: fused six-membered + five-membered heterocyclic rings of C and N o Bases: N-groups can accept protons, giving “basic” properties to the molecules o Need to know AGTCU, be able to distinguish the structures - “AnGels are PURe” - thymine is only in DNA, Uracil is only in RNA—you will never find them together - thymine has a CH3 group (only one that does) - It is always a purine + pyrimidine; A+T and C+G - Free bases are poorly soluble, and rarely occur - Interesting Nitrogenous bases (know these structures; be able to see the molecule and connect it to its function): o Theobromine and Theophylline: secondary metabolites of cocoa beans and tea leaves; act as a diuretic, cardiac stimulant, and vasodilator, relaxes smooth muscles o Caffeine: stimulant, diuretic; antagonist to adenosine adenosine is an inhibitory neurotransmitter that binds to the adenosine receptor; accumulates during wakefulness and helps promote sleepiness, especially following sleep deprivation - Nucleosides: nitrogenous bases attached to sugars o Glycosidic bond: between a nitrogenous base and sugar o 1’ attaches in sugar to Nitrogen o Rotation about glycosidic bond is possible - Common Ribonucleosides o Be able to recognize which are which o Purines end in “-dine” o If it is DNA, it is going to have “deoxy-“ prefix - Adenosine: o Inhibitory neurotransmitter synthesized in the brain, binds to adenosine receptors o Binding causes…. Drowsiness (slows down nerve cell activity) Dilation of blood vessels (lets more oxygen bind while sleeping o Clinically used as an anti-arrhythmic to slow/calm the heartbeats o Caffeine competes with the same receptor of the brain; reverses the effects of adenosine - Cordycepin (3’-Deoxy Adenosine) - o Antibiotic produced by Cordyceps militaris (scarlet caterpillar fungus) o Inhibits the final step of RNA biosynthesis by termination of the ribonucleotide chain o We do not make this, only made by the fungus o Also inhibits RNA synthesis of humans - Cytokinins: plant hormone derived from adenine; if you’re given the structure, identify the function o Contain adenine ring system with an attached 5-carbon hydrophobic group at the free NH2 o Ribose sugar o Promotes cell division in plants Zeatin Ribose: - Nucleotides: nucleosides with one or more phosphates - Common Ribonucleotides (recognize these structures): o TMP will not be found in nature because it is not found in RNA o You will never see deoxy UMP o IMP normally becomes AMP (it’s the transitional state before AMP occurs) - Common Deoxy-Ribonucleotides: - Metabolic functions of Nucleotides (besides building blocks of nucleic acids): o ATP: phosphate acceptor/donor; energy currency; deoxyATP is not used for energy because it can’t produce energy without oxygen o GTP: protein synthesis and signal transduction o CTP: membrane and storage lipid synthesis o UTP: carbohydrate synthesis and degradation - The Central Role of ATP in Energy Metabolism: o ATP allows us to do many energy-dependent things o Energy-dependent processes: Biosynthetic processes Active transport Mechanical work (muscles) Temperature regulation Bioluminescence - Closer look at the structure of Adenylate Nucleotides (ATP): o Energy is produced when phosphate bond is broken o Know types of bonds: Phosphoanhydride bonds are easily broken and that’s what produces energy Phosphoester bonds- alcohol group in the sugar interacts with the phosphate group Ester bonds- are found in fruit and it’s a mix of an acid and an alcohol (COO) - Cyclic Nucleotides o Second messengers in signal cascades o Second messenger: a TINY short-lived intracellular chemical signal molecule that relays a message (stimulus) from an external “first messenger”; this relay typically results in a cascade of events leading to a marked amplification of the first messenger o First messengers: hormones, neurotransmitters o Second messengers: Ca2+ ions, inositol-Pi3, diacylglycerol, cyclic nucleotides - How is cAMP formed? (cAMP is a secondary messenger) KNOW THIS: - Cyclic AMP and Cyclic GMP: secondary messengers o Adenylate Cyclase: ATP cAMP +PPi cAMP is involved in many signal cascades hormone signaling, apoptosis, disease reactions, neuron function o Guanylate Cyclase: GTP cGMP +PPi cGMP is involved in nitric oxide (NO) signaling blood pressure homeostasis, nerve impulse transmission, stress responses in plants (is a relatively new signal cascade- especially in plants) signals pain Biochem Lecture 8: - Formation of Phosphodiester bonds: DNA chain grows in the 5’-3’ direction o DNA is double stranded o Bond between phosphorous, and carbon-oxygen --- phosphoester bond (know difference between this and phosphodiester bond) o Bond connecting two nucleotides is a phosphodiester bond o Phosphodiester bond allows a long strand of nucleotides to form o Both ribose and deoxyribose form phosphodiester bonds o Misconception: in order to form a bond, you need a triphosphate o New chain addition joins from the bottom AKA 3’ end o Phosphate can only add at the 3’ end o 5’ is the first one in the line- has a phosphate at the end o First nucleotide has free 5’ end, last nucleotide has a 3’ end free o 3’ ends with an OH o When two strands form, must be antiparallel for DNA; basically flip the strand upside down so that 5’at the top now goes to the bottom - Formation of Phosphodiester linkage: o Formed with assistance from a TRIphosphate group o Must have triphosphate in it if it deals with DNA polymerase - Watson and Crick described the structure of DNA in 1953 o Figured out that phosphate groups had to face out so hydrogen bonds could be on the inside - B-DNA Spatial Dimensions o B-form of DNA is the form characterized by Watson & Crick. It was originally isolated from aqueous solutions as the partly hydrated “sodium salt”. It is thought that this form represents most native DNA in the cell. o Another form (the A-form) is observed when DNA is extracted/purified from ethanol, which tends to dehydrate the molecule and make it more compact than the B-form. Also, the angle of the base plane with the ribose plane in no longer perpendicular (as in B-form), but tilted 20 degrees away from perpendicular. o Major groove is about 2 nm across & 1 nm deep. This can easily accommodate a 0.5 nm alpha helix of protein. o Memorize: 3.4nm refers to the distance in one twist of DNA (twists about every 10 base pairs); so each nucleotide takes up about 0.34nm o Major groove and minor groove occur during each turn Major group interacts with proteins Minor group interact with drugs, small molecules, inorganic molecules o Helices are “right-handed” so they turn clockwise from each end o There is no linear space between base pairs - Stabilizing forces in DNA o DNA is relatively inert 1. Hydrogen bonds between base pairs: a. Two hydrogen bonds between AT and three between CG b. Harder to break bonds between C and G nucleotides 2. Hydrophobic interactions between bases a. Purine and pyrimidine bases are non-polar b. Bases attract each other towards the center of the double helix while repelling/excluding water; phosphates are at the surface attracted towards the water (much like lipids in a membrane) c. Excludes water 3. Electrostatic interactions: a. Sugar-phosphate “backbone” has negatively charged PO 43-groups b. Repulsions are avoided via shielding c. Charges improve solubility in water d. DNA is negatively charged because of the repeating phosphate groups - Functions of the Major Groove o Recognition sites for several transcription initiation factors o Specific domains of initiation factors lie in major groove o Promotes separation of DNA strands - Function of Minor Groove o Less is known about it o Often bind smaller (non-protein) ligands which then have several effects Inhibits some cancers- anti cancer drugs bind to this Antimicrobial activity - DNA Structure Varies: o Watson and Crick discovered B-DNA which is also known as sodium salt because it attracts positive charges o A-DNA: when RNA/DNA duplexes form because of low hydration o Z-DNA: zigzag formation as a result of torsion during transcription; left handed DNA - Erwin Chargaff (1950) o Found out that the concentration of A = concentration of T; concentration of G = concentration of C o Chargaff’s rule: A=T and C=G because they’re complimentary base pairs o All four (ATCG) should add up to 100 - Levels of Structure in DNA o DNA chain extended by DNA polymerase o Primary: the sequence of bases in a pair of complementary strands; just nucleotide complementary strands in double helix o Secondary: when it is completely coiled; can be linear (eukaryotes) or circular (prokaryotic chromosomes, plasmids, chloroplasts, mitochondria) o Tertiary: super-coiled DNA; must be super folded and coiled to fit in the nucleus - Packaging of Eukaryotic DNA o Bead and string model String= DNA Beads= Histones (proteins assisting in folding by having them wrap around) o 8 proteins of histones creates a nucleosome o histones are rich in basic amino acids (to make it really positive) because DNA is negatively charged o Chromatin: Heterochromatin: extremely condensed chromatin; completely inactive DNA in this form; no space between histones Euchromatin: active chromatin; nucleosomes aren’t as tightly packed and Linker DNA is exposed and able to be modified (transcribed); histones slide away - Chromosome packaging: A closer look at Nucleosomes o Nucleosome The basic unit of structure of eukaryotic chromosomes Composed of a core of 8 histone protein molecules wrapped with two coiled of DNA (~140 base pairs) plus ~60 base pairs of linker DNA between nucleosomes 8 different types of histones Histone core consists of 2 each of H2A, H2B, H3, and H4 protthns H1 (a 5 histone) facilitates higher level of coiling of chromatin; not considered part of the nucleosome In a bead and string model, there are 140 base pairs of DNA wrapped around the nucleosome, 60 base pairs of linker DNA; then another nucleosome How many nucleosomes are in 34,000 nm of DNA? Each base pair is 0.34nm 34,000/0.34 =100,000 100,000/200 (how many base pairs are in a nucleosome) = 500 nucleosomes Be able to manipulate base pairs and nm - Introduction to Ribonucleic Acids: o RNA is always single stranded and DNA is always double stranded - RNA vs. DNA o RNA is much more abundant than DNA because there are many more copies of RNA’s (messages, transfer species, ribosome numbers) made than the 2 copies of DNA. o Modified bases in tRNA include pseudouridine, dihydrouridine, 4-thiouridine, 1&2-methylguanosines especially in loops. o U is replaced by T (i.e. methylated uracil), because T will only base-pair with A; U, however is the only base that can base pair with any of the other bases. Evolutionarily, this could not be “tolerated” to maintain the fidelity required in DNA base pairing. o RNA sometimes folds over itself but then folds back o 5-10 times more RNA than DNA; why? Because DNA is the template for RNA and there is no limit to how much RNA can be produced with each template; RNA is more easily manipulated o Single-stranded nature of RNA allows it coil/fold back upon itself and nearby regions to base pair to form complex three-dimensional structures. Characteristic DNA RNA % of cell dry wt. 1 5 – 10 General structure Double-stranded Single-stranded* Sugar Deoxy-ribose Ribose Nitrogenous bases A,T,C,G A,U,C,G (+ modified bases) - Four Main Types of RNA: - tRNA, mRNA, rRNA, snRNA o snRNA’s thought to complex with proteins to form snRNPs (small nuclear ribonucleo proteins), are involved in removing introns and then splicing exons together to form functional mRNA’s. o snoRNA’s (small nucleolar) –snoRNA - small nucleolar RNA, forms snoRNPs, which process rRNA, mostly by methylation and isomerisation; thought to be involved in ribosome biogenesis. o siRNA - small interfering RNA, involved in gene silencing and regulation. o gRNA - guide RNA, needed for RNA editing, the removal and insertion of bases into mRNA. o tmRNA - an RNA molecule that disengages ribosomes from stalled translation of mRNA in bacteria. o telomerase RNA - an RNA molecule that forms much of the structure and all of the template required by telomerase. o hnRNA - rag-bag of unprocessed pre-mRNA transcripts and other heterogeneous nuclear RNAs of less well defined function. o The three most abundant are tRNA, mRNA, and rRNA (most abundant) - Transfer RNA (tRNA) o Flattened 2-D clover shape o ~75 nucleotides o tRNA’s are typically about 75 nucleotides long and have characteristic loops. o tRNA’s contain a variety of modified bases (e.g. pseudouridine, 4-thiouridine, 1-methylguanosine, dihydrouridine). o These (and other bases) consistently occur in the same positions of the tRNA molecule. o Each end/arm has a specific function: o 3’ end – amino acid attachment o o D loop (contains dihydro-uracil) – functions in aminoacyl- tRNA synthetase recognition. o TYC loop (contains pseudouridine) – functions in ribosome recognition binding. o Anticodon loop – functions in matching to the codon on mRNA during translation. o Typical shape is “clover-like” o An adapter molecule o Almost all have been manipulated from uracil o TYC arm- right arm for ribosome binding o D arm- left arm for RNA synthesis o Anticodon- matching the “triplet” codon on the mRNA during translation; the compliment of the mRNA goes on the anticodon arm in the 5’ to 3’ order; always corresponds to the codon o Everything is read 5’ to 3’ o The code is copied to form mRNA; each codon is 3 o 3’ end where amino acid is attached to tRNA, always end in ACC - Structure of Amino Acyl tRNA’s o Amino acids are linked to the 3’ –OH end of tRNA molecules by an ester bond Acid and OH make an ester bond - Functions of tRNA o Translation (protein synthesis): Carry (transfer) amino acids to ribosome for assembly into polypeptides At least one tRNA molecule for each 20 different amino acids Anticodon base pairs with mRNA codons Often described as an “adaptor molecule” - Ribosomal RNA o rRNA’s are structural elements of ribosomes (~60% RNA) o ribosomes contain a variety of RNA’s that differ primarily in their sedimentation characteristics (related to molecular weights) o larger the SU number, the heavier it is o Ribsomes are bigger and more complicated in eukaryotes - rRNA secondary “Road kill” structure is exceptionally complex o two different species can have almost identical shapes, but very different nucleotide sequences - Experiments leading to the identification of DNA as the genetic material o Griffith o Avery, McLeod, McCarthy o Hershey and Chase - Griffith’s experiment: some material gets transformed between bacteria o Rough Strain- didn’t have the capsule o Smooth Strain- had the capsule o Something in the heat killed the S strain - Avery, McLeod, McCarthy experiment: the material being transformed is DNA o Repeated treated four o Added an extra ingredient- added enzyme that killed the specific thing o Amylase- enzyme destroys all the CHO in that mix o Protease- destroyed protein o Added Dnase- digest DNA o Rnase- destroys RNA o Test which one is preventing transformation o DNase is not transforming - Griffith and Avery’s experiments o Non-virulent strain injected in mice and they live o Virulent strain injected into mice and they die o Heat killed virulent strain injected into mice and they live o Heat killed virulent strain and non-virulent strain injected in mice and they died - Hersey and Chase: their T4-Bacteriophage o DNA is hereditary material o Process a. Only the gens of a virus enter a host cell 1. start of infection 2. production of new virus particles 3. end of infection b. the virus’s capsid stays outside the cell - Viruses are tinier than bacteria - - First amino acid in all protein is methionine - Radioactive phosphorous would enter the bacteria - If proteins are genetic material they should find sulfur from the virus enter the bacteria or phosphorous - If centrifuge bacteria on the bottom and virus on the top - First case of centrifuging- P32 in the bacteria traveled with - Second: bacteria in the bottom and sulfur on the top so bacteria did not get the sulfur in the second experiment Lecture 9 notes: - I“formation Processing” Expression Transcription Translation DNA RNA Protein Replication - genetic engineering - - Variation in DNA structure: Cruciform DNA o Cruciform DNA: cross-like DNA structures that form when DNA contains a “palindrome” o Palindrome: a base sequence in DNA that provides the same information when read in either the forward or reverse directions - EcoR1 Restriction Site - DNA Gel Electrophoresis o Agarose o Ethidium Bromide o Larges piece on the top, smallest piece on the bottom - This information is for the gel below: o The fifth row is the known data o Each row uses the same plasmid but cuts it differently o More base pairs in the lines towards the top, so they’re brighter o You add the lines together to estimate the total number of base pairs o When it has a long block of base pairs, there’s either a lot of repeated palindromes or the DNA got degraded o In lane four, it only has one thick band because it got cut in half o Bigger bands on top, smaller on bottom - - Start and stop are the same point, goes clockwise - Cuts at each point where your enzyme appears - Should add up to 1000 every single time - Do not subtract - SANGER SEQUENCING: - To form a phosphodiester bond, you need the 3’ end of the sugar and the 5’ end of the incoming - dideoxyNTP - Common things in each sequencing: 1. Single stranded DNA template (5’ ATCGA 3’) 2. DNA polymerase (will add nucleotides to the sequencing strand) 3. deoxyNTPs (dATP, dTTP, dCTP, dGTP) 4. Unique for each lane! (added at 10x less than the total amount added; the A/G/C/T is what glows but this is relating to the complementary strand, not the template strand) o ddATP o ddGTP o ddCTP o ddTTP - Tube one: added from 5’ to 3’ end, and 10% of it will be ddATP and get stuck at A and DNA will not go any farther- will glow to show it stopped with 4 nucleotides - Tube two: will stop at G - Tube three: will stop at C - Tube four: will stop at T (does that twice for what we have) - If there are multiple A, T, C, or G in the complementary strand, it will have multiple lines for each time the reaction stops; just because the T is the first one and the last one in the strand, it doesn’t just produce an A* for the first T, it does it for both. - The lowest one appears at the 5’ end and the highest is the 3’ end - That gives you the complement so you need to find the complement base pairs in order to find the original DNA template strand - Builds up from the bottom and if you know what is in each lane; in order to find the original template strand in the 5’ to 3’ end, you will have to flip what you have found after finding the template strand (from 3’ to 5’) - Read it from bottom to top in the 5’ to 3’ end - DdNTP: lacks 3’ OH; does not let further nucleotides join; stops phosphodiester bonding after it is added - Complementary strand is the product of the sequencing reaction - All added into one lane and each nucleotide has a unique color o So everytime you see that color glowing, it represents a specific nucleotide - Variations in DNA Structure: Triple stranded DNA- a “Triple Helix” o Discovered in 1957 (after Watson and Crick) o Thought to form from partially unwound duplex DNA under “super-helical” conformational stress o Purines are double rings (A & G) while pyrimidines (T & C) are single rings. o Single ring pyrimidines are smaller, and more able to fit into major groove. o In addition to possible role in crossing over, triple helix DNA has been implicated in preventing DNA replication (ie. Triple stranded DNA can not be replicated), so this may have a role in preventing unwanted replication –eg. As in cancer). Another possible role may be associated with organization of nucleosome structure, because such triple stranded DNA can not be incorporated as part of nucleosomes. o If you have a stretch of purines on one side and pyrimidines on the other, the strands can separate and the pyrimidine wraps around the double stranded DNA forming a triple stranded DNA o The “third strand” occupies the major groove of the original duplex o Possible role in recombination (crossing over) - Genone Structure: Prokaryotes vs. Eukaryotes (Know the differences) o Plants have large genomes because of “polyploidy” duplications. Remember, most organisms are “diploid”, however, plants can be tetraploid, hexaploid, & octaploid. o Sugar cane=8n; oats=6n; alfalfa, peanut, tobacco, cotton all = 4n. o Repetitive DNA: segments that repeat often in multiple series. Approx. 45% of human DNA is repetitive. o Since prokaryotes are tiny, their genome size is muuuuch smaller than eukaryotes and their gene structure is much less complicated than eukaryotes; almost everything a prokaryote has, it uses/codes for something o 95% of the human genome doesn’t code for anything o For prokaryotes, they can control multiple genes with an operon; eukaryotes have an operator for each of the genes o Introns are not coded, exons are coding o Eukaryotes have repetitive DNA; each set of repetitions is unique to each person (like a signature); used in forensics o Pseudogenes were used by our ancestors but we no longer need; its just there - Transposons: o Discovered by Barbara McKlintock in 1940; found through corn kernal pigmentation Won a nobel prize in 1983 o Transposons= “molecular parasites” or “jumping genes” o Often 10’s-100’s of base pairs long o Large transposons can contain entire genes—transposable elements o Can cause mutations when inserted randomly in normal genes o “Alu” transposon family is best known in humans - Gene Structure Terminology: (KNOW THESE) o Operon: A group of linked genes that are regulated as a unit. o Plasmid: A small circular, self-replicating extra- chromosomal molecule of bacterial DNA. Plasmids are not normally required for host cell growth, but often confer “unique metabolic capabilities” and may become incorporated into host genome. o Introns: Non-coding (intervening) DNA sequences in eukaryotic genes. o Exons: Coding (expressed) DNA sequences in eukaryotic genes. o Pseudogenes: A non-functional sequence of DNA similar to a gene. Pseudogenes are likely remnants of a once- functional genes that accumulated mutations. o Transposons: DNA sequences that excise, replicate and insert themselves randomly elsewhere in the genome. Transposons are often referred to as “jumping genes”. - Plasmids are “small” (approx. 1000 to 400,000 bases). - “Unique Metabolic Capabilities” conferred by plasmids include the following: o Anitbiotic resistance o Virulence genes o Nitrogen fixation genes o Degradation of unique carbon sources (e.g. aromatic cpds). - Transposons are related to cancer; they are the remenants of viruses of our ancestors - Genome Segments from Different Organisms (be able to recognize the differences between each- mainly human and bacteria) o Notice the amounts of non-coding DNA in Humans & Corn vs. yeast & E. coli. o Also, notice the amounts of introns and especially the small amounts of coding specifically for genes in Human DNA. o Finally, Humans also have large amounts of DNA coding for tRNA’s. o Bacteria has no introns o Humans have a lot of introns o KNOW THE DIFFERENCES BETWEEN THESE o Focus on human vs. bacteria Lecture 10: DNA Replication General Characteristics of DNA Replication: chemically in the 5’ to 3’ direction semiconservative spacially bidirectional (has two forks at the same time) semidiscontinuous Duplicated genetic material must be completely duplicated before the mother cell can split into daughter cells Has to happen for every cell Replicates only in the 5 to 3 direction; nucleotides only added at the 3’ end (to the OH group) Messelson and Stahl: proved that DNA replication was semiconservative semiconservative: one strand of DNA is the original strand and the other strand is newly synthesized; conserves 50% and makes the other half from scratch Labeled proteins phosphorus or nitrogen (which is easier and not as detrimental to be around) and DNA sulfur Heavier DNA goes to the bottom, light DNA floats at the top Generation 0 will be labeled with heavy isotope (N15) If they grow with light nitrogen, and semiconservation occurs, after one generation, all will be hybrids Generation zero disappears completely after one generation and generation one becomes the new “parent” DNA Generation two will have half hybrid (N14/N15) and half would be light (N14/N14) Diluting out the N15 after every generation (so diluting out old DNA) At the end of 3 generations, 75% are light (N14) and 25% are hybrid no more heavy (N15/N15) For Conservative: no hybrids exist after one generation all either N15/N15 or N14/N14 NEVER SEE HYBRIDS EVERRR! Dispersive was never even really considered an actual idea Replication Origins Spacially replication is bidirectional Species share the same origins (where replication begins) Bacteria only have one origin DNA breaks open and replication goes in multiple directions both in the 5’ to 3’ direction Eukaryotes have multiple replication origins Where replication begins– replication bubble Replication fork is the V where DNA splits General Characteristics of DNA Replication: leading strand: one continuous motion that goes in the 5’ to 3’ direction lagging strand: multiple motions that go against the fork in order to be filled in the 5’ to 3’ direction; formed in little chunks as the fork exposes more DNA; small portions of a strand are called Okazaki fragments template DNA and polymerase on one strand repeatedly separate, reposition, and join together to resume replication of the lagging strand DNA polymerase can only work if an OH group is exposed Cannot combine two DEOXY nucleotides together Must have a “primer” (little stretch of DNA already involved in hydrogen bonding) in order to add more nucleotides through DNA polymerase Enzymes involved in DNA Replication: Helicases: break hydrogen bonds and split the double helix Single stranded binding protein: binds to the single strands after helicase breaks the hydrogen bonds DNA polymerase (3): fills out the new DNA in the 5’ to 3’ direction; must have primer RNA polymerase: can just bring in new nucleotides; does not need primer Primase: the enzyme that makes the primer; an RNA polymerase that produces RNA primers so new nucleotides can be added DNA polymerase (1): comes to replace the RNA with DNA Ligase: joins the okazaki fragments together to form a single strand of connected DNA Know what will happen if the enzyme is removed!! Steps of DNA synthesis 1. Initiation: unwind and stabilize the duplex DNA to form the replication fork a. Initiation Factors – (DnaA proteins) bind to origin of replication. b. Helicase – (DnaB protein) catalyzes the ATPdependent unwinding of duplex DNA c. Topoisomerases – prevent supercoiling & tangling of DNA during unwinding; bind ahead of the replication fork, nick supercoiling DNA, relaxes stress by allowing uncoiling. d. ssDNA Binding Protein – prevents reannealing of the separated single strands, protects against nuclease degradation. Specific names are not necessary to know Initiation factors (not enzymes) attract helicase towards it; a way to stop DNA from replicating; make sure a certain origin is visited my a helicase only once during a single replication phase Helicase has to have ATP to break hydrogen bonds!!; once it shows up, initiation factors leaves Topoisomerases: not near replication site; essentially prevent the unstable DNA from retwisting; untwisting it and making sure DNA does not fall apart; “supercoiling” 2. Elongation/DNA Synthesis: Replication: 5’ to 3’ synthesis of complementary DNA. a. Primase – an RNA Polymerase synthesizes a short (~ 10 nucleotide) RNA “primer”. i. DNA polymerases can not initiate DNA synthesis – they can only add nucleotides to the end of a chain that is basepaired with the template strand. ii. RNA polymerases can initiate synthesis without primer. b. DNA polymerase III – extends the RNAprimed chain. c. DNA polymerase I – later replaces RNA with DNA. d. DNA ligase – joins DNA (Okazaki) fragments. The DNA Polymerases in E. coli: DNA Polymerase I – (the “Arthur Kornberg Enzyme”) Fills in gaps, repairs miss matched pairs, replaces primer RNA during replication. DNA Polymerase II – Thought to also be involved in some repair processes; prevalent during stationary phase. DNA Polymerase III – The “main” polymerase of E. coli, (approx. 40 molecules per cell; Kcat – 1200/sec). It takes approx 1 hr. to replicate all the DNA in the cell in preparation for cell division. Primase: RNA polymerases are different because it can take two ribonucleotides and create a phosphodiester bond between them RNA polymerase can initiate synthesis without a primer 3. Termination: Not well understood. Ter binding proteins (in prokaryotes) bind to ter sites (~20 bp inverted repeat) on opposite side of DNA loop (bacterial chromosome), inhibit helicase and prevent further progression of replication forks. In eukaryotes, DNA polymerase runs off the ends of DNA; replication bubbles fuse as polymerases “collide”. Telomeres play a role in replication termination in eukaryotes (Telomeres protect the ends of DNA from being lost) Structure of DNA Polymerase Dimer: epsilon exonuclease repair function: Enzyme recognizes mispaired “bubbles” in DNA, backs up, excises, then polymerase resumes replication. theta subunit function is unknown. Multipurpose enzyme DNA polymerase (3) functions as a dimer; quaternary structure of the protein One monomer synthesize leading strand and one synthesizes the lagging strand at the same time Subunits to know ALPHA: forms polymerase reaction; BETA: sliding clamp; holds on to the DNA and moves down the DNA Epsilon: recognizes if there has been a mistake and has the ability to remove nucleotides in the reverse direction (in the 3 to 5 direction); exonuclease; “proof reading” 1 nucleotide per 1000 nucleotides= the mistake rate Many checkpoints/ stages to correct mistakes and it starts with DNA polymerase III Subunits of DNA polymerase III: Processivity is the frequency with which an enzyme dissociates from the template during DNA replication. Know the ones in the boxes Beta is part of the core Overview of E. coli “Replisome It’s a dimer so the leading and lagging strands are produced at the same rate/time The lagging strand flips around so the lagging and the leading strands can both go through the DNA polymerase at the same time Enzymes at the fork: helicase, primase, single stranded binding protein, DNA poly 3 dimer; REPLISOME: anything found at the replication fork Primosome complex: primase and helicase as soon as the helicase opens the DNA, primase acts on it DNA ligase, DNA poly 1, and topoisomerase NOT in the replisome The Ends of DNA are not Completely Replicated: Telomeres are at the end of chromosomes and they are not coded Everytime we replicate our DNA(every 24 hours), the ends of the DNA do not get replicated and therefore it chops off; telomeres are found at the end so they are what disappear after every replication Telomerase is not produced as much as you get old Telomere Structure and Function telomere: a series of repeated TTAGGG DNA sequences located at the ends of linear eukaryotic chromosomes Each time a cell divides (and replicates its DNA), some of the telomere is lost due to exonuclease activity (i.e. ends are degraded). Eventually little or no telomere remains, but degradation continues, and the cell dies because “vital” genetic information is lost. Thought to be part of the aging / senescence process. Telomeres and cloning: premature aging and death of clones (“Dolly” the sheep) thought to be related to cloning from cells/nuclei with partly reduced telomeres telomerase: an enzyme that restores (synthesizes) telomere sequence thought to restore chromosomes and cell longevity some cancers may be due to overactive telomerases which gives “immortality” to chromosomes/cells/cancers In 1961 American biologist Leonard Hayflick discovered the principle of cell divisibility. The most important implication of this principle is that “critical” cells in human organisms (i.e. brain, heart, the nervous system) cannot divide indefinitely. The maximal number of cell divisions is on average 50±10 (the so called Hayflick’s Limit). Some scientists believe that the loss of a cell’s ability to reproduce is a major cause of aging. Telomerase Function: comes in with RNA primer that sits at the end and extends the end; exposes a free 3’ end so DNA polymerase fills in the missing DNA Summary Comparing DNA Replication in Prokaryotes vs. Eukaryotes proteins are similar, except that prokaryotes only have one origin of replication and eukaryotes have multiple prokaryotes replicate faster Polymerase Chain Reaction (PCR) Replication in a test tube Increase the genome content amplifying a sample Take a piece of double stranded DNA, heat it to 95 degrees C to denature the DNA (doesn’t destroy the DNA), cool the DNA at 45 so primers come stick, heat again to 72 degrees so it can be extended Every copy doubles after just one round of PCR INGREDIENTS: 1. Template DNA 2. Primers to join at the ends of the template strands 3. Deoxyribose nucleotides (dNTPs) 4. DNA polymerase III (can survive at really high temps)
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