March 28-April 1 Notes
March 28-April 1 Notes CELL 2050
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Joseph Merritt Ramsey
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This 10 page Class Notes was uploaded by Joseph Merritt Ramsey on Monday April 4, 2016. The Class Notes belongs to CELL 2050 at Tulane University taught by Dr. Meenakshi Vijayaraghavan in Winter 2016. Since its upload, it has received 23 views. For similar materials see Genetics in Science at Tulane University.
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Date Created: 04/04/16
April 1, 2016 Chapter 12: Gene Transcription and RNA Modification Overview of Transcription o Only time that DNA can be accessed for the information to be expressed o Players Involved 1) DNA – codes for RNA, has signals for starting/stopping 2) Proteins – recognize, regulate, modify transcribed RNA o DNA does not change through the process – a copy is made identical to one of the strands o What is a Gene? A unit of heredity DNA sequences that codes A transcriptional unit (not necessarily structural) o Some Terminology o Central Dogma DNA RNA Proteins Classes 1. Eukaryotes o Dogma follows, and Polycistronic RNA cannot occur because organelles perform the specific tasks 2. Prokaryotes o Can have Polycistronic RNA – can code for several types of proteins Consider, a given vitamin or mineral needs to be processed In Prokaryotes, a single long strand of mRNA can be transcribed for various proteins in the metabolic pathway o This can occur in mitochondria as well due to their evolutionary nature 3. Outliers o Viruses are organisms with RNA as their genetic material o This is important to note that the RNA in viruses does not act like a protein, can only perform the same functions o Viroids are infectious RNA’s, but they only affect plants (no domains) o Prions are infectious Proteins that unbind other proteins, more complex than simply RNA, because they have domains Components of Transcription o Transcription Sequences DNA and RNA each have specific sequences needed for Transcription DNA RNA Starting Starting o Promoter site for RNA o Prokaryotes Polymerase Binding Ribosomal Binding Site Stopping Start Codon – specifies the first o Terminator Sequence amino acid in the sequence, modified AUG in Prokaryotes Regulatory o Eukaryotes o Also has regulatory sequences that alter and Have 3’ and 5’ Modifications, so influence the rates of just a start Codon is needed (AUG) transcription (different than Prokaryotes) The Ribosome will travel back and forth over the mRNA before it “decides” which start codon Stopping o o Transcription Steps I. Initiation The strands have to separate to be accessible o Done by transcription factors and RNA Polymerase They bind and a Complex Forms II. Elongation The actual sequence is constructed III. Termination Process stopped Prokaryotes have Rho Dependent and Rho Independent Pathways Occur when the DNA/RNA Hybrid is broken (separated) o This is the temporary connection between the Template and the mRNA o The Various Roles of RNA Transcripts – This is an important point to consider: RNA has numerous functions it can perform after transcription 1. Structural This is mRNA Codes for proteins, over 90% of genes 2. Standard tRNA and rRNA are also present in high concentrations Involved in the Transcribing portion 3. Atypical (Assessed by the Sedimentation Coefficient) 7S is the Signal Recognition Particle (SRP) (7S + 6 Proteins, co-translational recruitment) Spliceosomes (snRNA and snoRNA) Nucleolus RNA Viruses Steps Detailed o I. Initiation A. Promoter Sequence +1 Location on the DNA Has TTGACG at -35 and TATAAT at -10 crucial for the +1 location o Binding first occurs on the -35 sequence o 16-18 bp long o Upstream Promoter sequences essentially “point” to the +1 Location Alteration in the binding sequences results in the rate changing from B. RNA Polymerase II (Holo-Enzyme, made of two Core Enzyme compenents) Subunits: Has a Motif (super-secondary structure) that binds to - 35 location on the major groove Core Unit Various Subunit Components β (and Beta Prime) – o β and β’ catalyze ester formation in both Catalyzes the bond Eukaryotes and Prokaryotes ω – Assembles the Core o ω – Assembles the Core σ Factor o Single subunit that allows the Core to function o Induces the opening up of the strands C. Haloenzyme Binding It binds loosely and beings to “scan” the DNA for a Promoter Sequence Then the Closed Complex forms o This occurs when the σ Factor and DNA Poly II bind at the -35 Promoter Region Shortly afterwards, the Open Complex Forms o The strain placed on the molecule from the Closed Complex causes the adjacent AT rich region to unravel, forming the Open Complex D. Short RNA Strand Synthesis In the Open Complex, a small strand of mRNA is formed (8-10bp) This synthesis causes the release of the σ Factor and the End of Initiation The Core Enzyme continues to move and transcribe o II. Elongation The actual sequence is constructed in this step The Transcription Bubble forms as copying occurs About 17bp long Codes at a rate of 43 Nucleotides Hz o III. Termination This is marked by the breaking of the DNA/RNA hybrid Prokaryotes rely on Rho Pathways Rho Dependent Rho Independent Rho acts like Helicase Intrinsically occur Has Requirements to Bind because of the nature o Rho Utilization Sequence (Rut) in DNA of the mRNA sequences o Alternating GC Rich Regions to form Stem Loop Process o Stem Loop forms downstream o This occurs because of the GC Rich Alternating Regions o They have an affinity for each other o The Stem Loop slows down polymerase by blocking the Ribosome o So Rho can catch up from behind March 30, 2016 Chapter 11: DNA Replication Chargaff’s Rule is Important to Consider Discovery of the Process Bacterial Replication The Overall Process o 1. Prepping A. Methylation B. DnaA-Box/ATP Complex Proteins C. DNA Helicase D. Single Strand Binding Protein E. Gyrase (Topoisomerase II) o 2. Synthesizing A. DNA Primase B. DNA Polymerase II, IV, and V have repair functions I and III have normal replication functions o 3. Polymerase Action DNA Polymerase Actors A. Polymerase III B. Polymerase I Reaction of DNA Polymerase -Subunit Importance of the β-Subunit o 4. DNA Ligase Acts about every 500,000bp, When Poly III Hops off Prokaryotes use NAD , Eukaryotes ATP o 5. Termination But the Ter Sequence also plays a role Tus binds to the Ter Sequence and triggers termination Ligase comes in after the Tus initiated termination Sometimes Ligase has errors, but Topoisomerase II comes in to help fix Catenanes DNA Replication Complexes (they form to increase efficiency) o 1. Primosome (H-Bonds and Primers) o 2. Replisome (With Poly III) o 3. DNA Polymerase III Unit Proofreading Mechanisms o Fidelity – retaining the correct DNA sequence I. Inherent Instability of Mismatched Bases II. -Subunit Specificity III. Εpsilon-Subunit has a Proofreading Function How Does the Cell Coordinate Replication with Division? – Components are Needed o 1. Methylation (Adenine Mutation) - Results in Hemimethylated strands o 2. DNAa-Box Proteins o 3. Cell Growth and the Dam Gene Confirmation of Triphosphate Theory – Arthur Kornberg o He theorized the Triphosphates were recruited to form Nucleotides o Set Up and Procedure Took all the necessary proteins and components to necessary to synthesize (Complete System) as well as a DNA template Add the end of the replication period (30min), Perchloric Acid was added to destroy the remaining bases The centrifuged and took the DNA Pellet o Findings Complete the template, fεound radioactivity present ( P) Without the template present, all nucleotides broken down and no radioactivity was present Mutants and Quest for Discovering Protein Function o How could the function of various replication proteins be discovered and determined? If mutants were observed, the functionality could be determined Allowed them to discover DNA Polymerase II and III in Bacteria o Conditional Mutants The mutations had to be screened One by one, Brute Force Screening to what mutations were present They couldn’t induce a specific locus to mutate Specifically observed a particular type that were sensitive to the environment (Temperature Sensitive, ts) These would act in Permissive Temperatures o Experimental Procedure I. Mutagenic Agent (usually Radiation) II. Agar Plating III. RepliPlating They touch the bacterial colony onto a disk They then grow that colony and simultaneously touch two other plates in order to replicate IV. Permissive vs. Non- Permissive Temperature o Experimental Findings Rapid Stop Slow Stop Vital to Replication Important in the Second process, causes Round, so Replication immediate stop halts after one go Polymerase I,III, Not enough DnaA-Box Helicase, Primase Proteins All are vital to DnaC cannot recruit Replication Helicase Dam does not function properly o Importance of Understanding 1) Identification of Proteins Function 2) Mapping of Given Genes 3) Genetic Cloning (cDNA) could now Occur Eukaryotic Replication Overview o Prokaryotic Comparisons (Highlight Similarities in Replication Processes) 1. OriC Locations – one in prokaryotes, various in Eukaryotes (every 100,000bp) 2. DNA Nature – linear with histones vs. naked circular 3. Cell Cycle Regulation Checkpoints – protein checkpoints to push forward, complex methods o Still utilize many of the same proteins as well: 1. Gyrase 2. Single Stranded Binding Proteins 3. A form of Primase Process o I. Prepping 1) Origin of Replication (ARS) 50bp Long in Eukaryotes Known as Autonomously Replicating Sequences (ARS) Element Contain: o 1] These sequences have AT-Rich Regions in them o 2] They also have a protein binding sequence analogous to DnaA-Box Regions 2) Pre-Replication Complex (with the Origin Recognition Complex) The PreRC is made of 14 Proteins This occurs during the G1 Phase A particular complex within the PreRC determines initial recognition o Origin Recognition Complex (ORC) o Made of 6 Proteins o Forms an ATP complex and binds to ARS (analogous to DnaA-Proteins) This binding initiates the S Phase (leads to MCM) 3) MCM Helicase MCM Helicase initiates the replication process (this is the beginning of S Phase) This first break is in the AT Regions of the ARS MCM Helicase must be present to form replication forks (wherever it is present, replication forks form) o II. Primer Formation 4) Polymerase- (with Primase) Works in conjunction with Primase to make DNA Complex Forms a 10bp RNA Primer and 20-30bp DNA Strand This is the Primer Stage Theory is that only has a 20-30bp capacity o III. Synthesizing 5) Secondary Polymerase Action After the Alpha/Primer Complex has acted, Alpha falls off and either δ (Delta) or ε (Epsilon) Leading o ε works in the leading strand Lagging o δ works in the lagging strand o Needs to have a motile capacity in order to function in Lagging Strand o Works on 100-200bp Long Fragments (more origins so no needs for long strands) 6) Primer Removal DNA Polymerase I worked in Prokaryotes o In Eukaryotes, δ-Polymerase works always, leading and lagging, with Flap Endonuclease Works by creating peripheral “flaps” that can be removed Lagging Process o I. Moves up the fragment and “butts into” and “pushes off” a 2bp Long fragment (the next primer upfield) This process occurs on the whole primer o II. Flap Endonuclease comes and removes the small strand o III. Delta then continues to add DNA o IV. As a longer strand accumulates, DNA Helicase (DNAII) comes in and removes (5+bp) Leading Process o I. PCNA (Proliferating Cell Nuclear Antigen) allows for Delta Polymerase separation and reattachment Analogous to β Clamp in Prokaryote Polymerase III This proved processivity to allow it to work Also present for Epsilon, but the notes action is the on and off falling of Delta o II. Then the above process occurs on the single location in the leading strand Polymerase Types δ ε γ Also the Replaces Replaces Replication of catalyzing type Alpha in Alpha in the Mitochondrial Works with lagging strand Leading DNA Strand Primase to Removes form initial Primer primer Repair o Types – Works in conjunction with cell checkpoints (such as p53) I. Polymerase β can remove bp mismatches Base excision repair – removes incorrect bases to be replaced Works as replication is occurring II. Lesion Replicating Polymerases Continues elongation if repair does not end up occurring o Involved in continuing replication of replication Interestingly, can also detect mismatched bp in the parent strand and allows for a correction in the daughter without altering daughter o Recognition 1. H-Bonding Mismatch 2. Induced Fit and DNA Alignment 3. DNA Tortion Nucleosome Replication o Histone need to be replicated as well (H2A, H2B, H3, H4 from the Octamer especially) o Also follows a Semiconservative Model (so old and new histones form) Telomere Replication o When the Delta Polymerase comes to the end, what happens with the Telomeres? The loops are 100-200bp, so what if the remaining end is not exact? The template isn’t long enough – 3’ Overhang remains Telomerase acts (loses functionality with age, so Telomere length reduction) o Telomeres The remaining part is known as the 3’ Overhang These are usually about 12-16bp Long Telomeres have numerous Repeat Sequences comprised of GGG and TT Repeats Normal Polymerase function presents some problems: Cannot flip/loop Still has to code 5’ to 3’ o Telomerase Process TERTS (Telomere Reverse transcriptase) is a part of Telmoerase There are two of these proteins present RNA is used as a primer to extend Template Strand TERTS has an internal 9bp RNA Complementary Strand that codes a DNA fragment Attaches at first three Codes six Repeats It is then moved to the 3’ end since replication and looping cannot occur there Ligase stiches the translocated portion together Forming a template long enough to form a loop which can then be replicated