Final Study Guide
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Date Created: 04/30/15
LECTURE FOUR STUDY GUIDE How do bacteria sustain life on earth 0 Provide oxygen we breath fix nitrogen decompose carbon Why are bacteria good model organisms 0 They are easy to work with they can be grown on different media they are easy to genetically manipulate On an evolutionary tree what indicates common evolutionary ancestors 0 Nodes List the three organisms that aren t microbes 0 Animals fungi and plants Which organisms are prolltaryotes 0 Bacteria and archaea True or false prolltaryotes do not have a nucleus 0 True True or false eulltaryotes have a nucleus 0 True What is a nucleoid 0 Single circular bacterial chromosome not bound by any membranes Do bacteria have organelles 0 No What is the unique cell wall of bacteria called 0 Peptidoglycan What part of the bacteria do antibiotics attack 0 Peptidocglycan layer Describe plasmid 0 Circular piece of DNA that carries nonessential information How do plasmids move 0 Via flagellum through liquid or a hard surface What do plasmids often time do 0 Give extra phenotype What is a capsule 0 Sticky substance on the outside of a bacterium How do capsules decrease chances of immune system attack 0 They act as a mask from the immune system Eulltaryotes are large bacteria are 0 Small Prolltaryotes have a nucleoid eulltaryotes have 0 A nucleus If eulltaryotes have membranebound organelles prolltaryotes have 0 None If prolltaryotes are 70S ribosomes what size of eulltaryotes o 805 Which organisms do not have a cell wall 0 Animals Which organisms have a cellulose wall 0 Plants and fungi Which organisms have peptidoglycan 0 Prolltaryotes If eulltaryotes have a linear chromosome what do bacteria have 0 Circular chromosomes What does it mean to have a diverse metabolism 0 The ability to grow essentially everywhere What does it mean to be an extremophile 0 The ability to grow essentially everywhere How are bacteria important in the carbon cycle 0 They cycle carbon I How 0 Convert C02 to sugars What importance do bacteria have regarding oxygen 0 Cyanobacteria in the ocean provide much of the oxygen that we breath What importance do bacteria have regarding nitrogen 0 Fix nitrogen in the plant root in a form that the plant can use Which part of bacteria is responsible for pathogenicity 0 Capsule When Fredrick Griffith injected S strain into a mouse what happened 0 It died When Fredrick Griffith injected R strain into a mouse what happened 0 It lived When Fredrick Griffith injected heat killed S strain into a mouse what happened 0 It lived When Fredrick Griffith injected heat killed S strain and living R strain into a mouse what happened 0 It died What did Avery s experiments based off of Griffith s experiments prove 0 Nucleic acids are the hereditary material Define bacteriophage 0 Viruses that infect bacteria Generally describe the components of bacteriophages 0 Genome DNA or RNA capsid head protein that surrounds the genome and sometimes a tail Describe the lytic cycle five steps 1 Phage infects bacterium and injects its genome 2 DNA is replicated 3 Phage particles are produced 4 Bacterium bursts 5 Phage is released Describe the lysogenic cycle four steps 0 1 Phage infects bacterium and injects its genome 0 2 Phage DNA integrates into bacterial chromosome prophage 0 3 indefinite replication n of prophage 0 4 induced to switch to lytic cycle What did Hershey and Chase use to confirm DNA is the hereditary material 0 Bacteriophages What did Hershey and Chase observe that led to the conclusion that transforming agent is nucleic acid 0 DNA was injected not proteins What DNA structure is the result of it twisting 0 Major and minor groove Which proteins interact with DNA at the major groove 0 DNA binding proteins Which proteins interact with DNA at the minor groove 0 Histone proteins Describe the parts of a nucleotide 0 Nitrogenous purine pyrmidine base sugar deoxyribosome and a phosphate List the two bases that are pyrimidines 0 Cytosine and thymine OOOOO List the two bases that are purines 0 Adenines and guanine What is Chargaff s Rule 0 The amount of QC pairing or AT pairing might differ but purinepyrmidine ratio will always be approximately 11 Where does a phosphodiester bond form in DNA 0 Between 5 phosphate and 3 hydroxyl How many hydrogen bonds form in an AT bond 0 Two How many hydrogen bonds form in an GzC bond 0 Three Describe what makes DNA strands antiparallel 0 Phosphodiester bonds in DNA What conditions cause DNA to denature 0 High temperatures or high pH What determines the melting temperature of DNA 0 CC content and ionic strength of solution the higher GzC content the higher the temperature LECTURE EIGHT STUDY GUIDE Define dispersive model 0 Parental strands broken into double stranded fragments and use as templates Define semiconservative model 0 Each parental strand serves as a template Define conservative model 0 Parental strands remain together What was the goal of the Meselson Stahl experiment 0 Determine the mechanism for DNA replication What did the MeselsonStahl experiment do 0 Grow bacteria containing one of two different isotopes of nitrogen heavy or light Why did the MeselsonStahl experiment use nitrogen 0 Because each of the four bases contains nitrogen How was DNA separated in the Meselson Stahl experiment 0 By density Define origins of replication 0 Sequence specific positions where replication is initiated Define prereplication complex 0 Complex of several proteins that locally melt the double helix to create replication bubbles How many replication forllts does a replication bubble have 0 Two What unwinds DNA 0 DNA helicase What synthesizes primers 0 Primase Which direction does replication occur 0 5 I 3 direction Define leading strand 0 Strand that is being synthesized in one long continuous stretch Define lagging strand 0 Strand that is synthesized in several short bursts Why does the lagging strand lag 0 Unwinding of DNA must occur first so that there is available 5 so that it can be synthesized What does topoisomerase 0 Relieves structural tension How does structural tension arise in DNA 0 Unwinding of dsDNA puts strain on unwound DNA Where do positive supercoils accumulate 0 In front of the replication fork Define topoisomerase 0 Protein that acts ahead of the replication fork and breaks one or both strands of DNA How does topoisomerase relieve structural tension 0 By breaking DNA passing NDA through the break and putting the ends back together What do ssDNAbinding protein do 0 Bind newly separate strands to keep ssDNA from reannealing to dsDNA What does primase bind to 0 ssDNA Once primase binds to ssDNA what does it do 0 Add a short primer Define primer 0 DNA RN A sequence that is complementary but shorter than the template it will be removed at the end of replication Why is a primer necessary 0 It serves as the starting point that DNA polymerase recognizes List the three domains of DNA polymerase 0 Palm fingers and thumb Define the palm domain of DNA polymerase 0 This catalyzes DNA synthesis and monitors base pairing Which part of DNA polymerase are the nucleotides added 0 Palm Define fingers 0 This bends the template strand positions next base to be pair Define thumb 0 Maintains association between DNA polymerase and DNA LECTURE NINE STUDY GUIDE In DNA synthesis what is added to the growing DNA strand 0 dNTP a base How are phosphodiester bonds formed 0 Removal of beta and gamma phosphates Where does the phosphodiester bond form 0 Between 3 OH and alpha phosphate of incoming dNTP Define DNA polymerase processivity 0 The ability to add many dNTPs before falling off the template strand Define exonuclease 0 A molecule that proofreads synthesized DNA to remove improperly basepaired dNTPs Adding an incorrect nucleotide does what 0 Alters position of 3OH I Which leads to 0 Reduced rate of DNA synthesis 0 Why I Because the end is no longer in the proper orientation for the active site In removing of a mismatched nucleotide the primertemplate junction is destabilized which leads to 0 DNA becoming single stranded I Which leads to 0 Lower affinity to the palm What has high affinity to the exonuclease active site 0 Singlestranded DNA What happens to singlestranded DNA once it is in the active site 0 The incorrect nucleotides are removed What increases DNA polymerase processivity 0 Sliding clamp Where does the sliding clamp bind 0 DNA polymerase How does the sliding clamp keep DNA polymerase from diffusing away 0 Interaction between sliding clamp and DNA polymerase is stronger than the interaction between DNA polymerase and DNA When does DNA polymerase lose affinity for sliding clamp 0 When DNA polymerase has doublestranded DNA in its active site How many base pairs does primase synthesize 0 Approximately 10 Does primase have high or low processivity 0 Low I Why 0 Does not interact with sliding clamp Define holoenzyme 0 A structure that physically linllts three DNA polymerase and one sliding clamp loader In the holoenzyme which orientation do the DNA polymerases have 0 Opposite direction I Why I To allow for 5 3 synthesis to occur on antiparallel strands Define replisome 0 All proteins working together at the replication fork Define RNaseH 0 An RNA primer that removes much of the RNA primer Define endonuclease 0 This removes nucleotides internal to DNA How does RNaseH cleave nucleotides within the primer 0 Cleaves between RNA nucleotides What removes the last RNA nucleotide 0 Exonuclease I Why not RNaseH 0 Because the last RNA nucleotide is by a DNA nucleotide and RNaseH can t uncleave this bond What does DNA polymerase recognize 0 Primerztemplate junction What does DNA ligase do 0 Fill in the nicllt by forming the final phosphodiester bond in the strand and connecting the nucleotides Where are telomeres 0 Ends of linear chromosomes Describe telomeres 0 A and T rich sequences repeated at the end of the chromosome Which strand has an end replication problem 0 Lagging strand Why does the lagging strand have an end replication problem 0 DNA polymerase can t fill in the end of the chromosome I How does telomerase solve this problem I It replicates the ends of linear chromosomes How does telomerase replicate the ends of linear chromosomes 0 Binds to lagging strand and adds bases complementary to its own RNA What is Werner syndrome 0 A disease caused by a WRN mutation WRN encodes helicaselillte proteins That leads to a shorter life span I Why does Werner lead to a shorter lifespan 0 WRN gene mutation is implicated in accelerated telomere shortening and in decreased telomerase LECTURE ELEVEN STUDY GUIDE How many origins of replication do prolltaryotes have 0 One Can an origin of replication for eulltaryotes initiate replication more than once per cell cycle 0 NO only prolltaryotes can do this In DNA synthesis what is added to the growing DNA strand How many origins of replication do eulltaryotes have 0 Multiple only one is activated per cell cycle What drives initiation of DNA replication in prokaryotes E Coli 0 ProteinDNA and proteinprotein interactions How do I describe the initiation of DNA replication in E Coli How does a prolltaryotic cell divide faster than replicate 0 Use multiple replication forks In using of multiple replication forks does replication occur successively 0 No new rounds of replication can begin before the old round ends What direction is replication 0 Bi directional When is the entire genome replicated 0 Before cell division When is the prolltaryotic chromosome actively replicated 0 When the chromosome is segregated How many times does the eukaryotic chromosome replicate 0 Once Do eukaryotic cells divide while the chromosome is being replicated 0 No LECTURE FIFTEEN STUDY GUIDE What are the DNA bases 0 Adenine guanine cytosine and tyrosine What are the RNA bases 0 Adenine guanine cytosine and uracil What is DNA sugar 0 Deoxyribosome What is RNA sugar 0 Ribose What is the difference between DNA and RNA sugar 0 Ribose has a 2 OH and deoxyribose doesn t How are RNA nucleotides linked together 0 Phosphodiester bonds Where are RNA phosphodiester bonds formed 0 Between 3 OH of one ribonucleotide and 5 phosphate of another RNA is almost always double or single stranded 0 Single stranded How does RNA form a secondary structure 0 By folding back on itself List the four secondary structures of RNA 0 Stemloop structure hairpin internal loops bulge junctions Describe the stemloop structure 0 Short stretches of complementary sequences base pairing Describe internal loops 0 Unpaired nucleotides on either side of the stem Define bulge 0 Unpaired RNA nucleotides on one side Define junctions 0 Site where three or four stems branch off DNA is into messenger mRNA 0 Transcribed Define transcript 0 Product of transcription What does the transcript act as 0 Message or a set of instructions for making protein Define ribosomal RNA 0 RNA that forms the structure of ribosome recognizes messages and assembles proteins Define transfer RNA 0 RNA that activates amino acids and reads the mRN A message Define messenger RNA 0 RNA carries the message from the DNA What does RNA polymerase do 0 Catalyze the synthesis of RNA Does RNA polymerase require a primer 0 No What does RNA polymerase use as a template 0 One strand of DNA Which direction does synthesis of a new DNA strand by RNA polymerase occur 0 5 I 3 direction What is the coding strand of DNA 0 Nontemplate strand What is the template strand of DNA 0 The strand RNA polymerase uses to make the complementary RNA sequence How many transcripts can be transcribed from a single gene 0 Multiple What is the purpose of replication 0 Copy the genome once per cell division What is the purpose of transcription 0 To selectively copy sections of the genome and make one to thousands of copies of that section LECTURES SIXTEEN STUDY GUIDE What are the three steps of transcription initiation 0 1 binding of RNA pol to a closed complex promoter promoter is doublestranded 0 2 DNA strands separate to form an open complex 0 3 Initial transcription promoter escape What are the five subunits of the RNA polymerase core 0 OiI Alpha one XII Alpha two Beta prime 5 Beta Q Omega Where is the active site in RNA polymerase 0 Cleft of the claw What does the sigma factor do 0 Determines which promoters RNA polymerase will bind to Define sigma factors 0 Proteins that bind specific promoter sequences and instruct RNA polymerase where to begin transcription Define sigma70 0 Aka housekeeping sigma this sigma factor directs transcription of most genes in cell genes with essential or routine function Where does sigma70 bind 0 Promoters with 35 and 10 regions Does sigma 70 recognize a consensus sequence or a specific sequence 0 Consensus sequence If a promoter is closer to consensus it means 0 That genes need expression at higher levels If a promoter is far from consensus it means 0 That the genes do not need expression at higher levels Define isomerization 0 Formation of a closed complex Which sigma factor produces an open complex 0 Sigma 2 I How 0 By recognizing and melting the 10 region When does sigma 2 melt the 10 region 0 When two bases A11 and T7in the nontemplate strand flip out and insert in pockets with 02 Define abortive synthesis 0 RN Ap stalling at the promoter and only producing releasing numerous short transcripts Define transient excursion 0 A mechanism of initial transcription in which RNAp moves forward to synthesize and release RNA and then retreats along the template Define inchworming 0 A mechanism of initial transcription in which a flexible region allows the front of RNAp to move downstream to synthesize and release RNA and then retract Define scrunching 0 A mechanism of initial transcription in which RNAp remains stationary and bound to promoter as it pulls DNA into itself Why does promoter escape occur 0 Because RN Ap is still stuck in promoter escape During promoter escape which interactions are broken 0 Polymerasepromoter and coresigma factor interactions are broken Where does the energy from promoter escape come from 0 Scrunching mechanism What happens after promoter escape 0 Elongation LECTURE SEVENTEEN STUDY GUIDE How many hydrogen bonds are formed between an adenineuracil thymine bond 0 Two How many hydrogen bonds are formed between a guaninecytosine bond 0 Three Describe the chemical reaction of RNA synthesis much like DNA synthesis 0 Beta and gamma phosphates are removed from incoming ribonucelotides a phosphodiester bond is formed between previously added NTP and incoming NTP to form the backbone In transcript elongation what happens to the RNA that has been translated 0 It passes through the RNA exit channel How does RNAp advance 0 It uses a step mechanism NOT SCRUNCHING to advance one base pair for every nucleotide that is added to the transcript Name the mechanisms RNA polymerase uses for proofreading 0 Pyrophosphorolytic and hydrolytic editing Define pyrophosphorlytic editing 0 Removing incorrectly inserted ribonucleotide by adding beta and gamma phosphates back on the NTP I This is a reverse of which reaction 0 Synthesis After pyrophosphorlytic editing occurs what happens 0 The correct ribonucleotide is inserted in its place Define hydrolytic editing 0 RN Ap bacllttracllts one or more nucleotides cleaves RNA and then removes sequence with the error Name the mechanisms used to terminate transcription 0 Rhoindependent termination and Rhodependent termination Define rhoindependent termination 0 A transcription termination that requires formation of a CG stem loop Define rhodependent termination 0 A transcription termination that requires protein Rho to terminate transcription Describe the sequence of Rhoindependent terminator 0 Short inverted repeats separated by 8 AT enriched base pairs followed by a string of T s What happens once the Rhoindependent terminator is transcribed 0 It forms a stable stemloop structure What dos the stem loop structure do 0 Knock off RNA polymerase from DNA template and halt transcription Define RHO 0 A protein made of six copies of one subunit that binds to mRN A at a rut site Define Rut sites 0 Rho utilization sites where RHO binds to mRN A made of C that occurs approximately 12 bases How doe RHO move along the RNA transcript 0 Using DNA Describe RHO s helicase activity 0 The unwinding of RN A DNA hybrid which causes RN Ap to dissociate Define gene 0 A segment of DNA that codes for a functional product protein Define the central dogma 0 DNA I transcribed I mRN A which is I translated lint protein Each filament is made of copies of 0 Flagellin Define flagella filament 0 Rotates to provide propulsion made of copies of flagellin Describe hoollt of the filament 0 Flexible joint Describe basal body of the filament 0 Connects via flagellum to the cell body and harnesses power for rotation governs flagellar biosynthesis 0 Fla Che operon CHAPTER EIGHTEEN STUDY GUIDEB Define transcriptional mechanism 0 Mechanism by which gene expression is increased or decreased in response to the environment Define activation 0 Increase of expression by the environmental input Define repression 0 Decrease expression by the environmental input What do alternative sigma factors do 0 Replace sigma70 with other subunits Different sigma factors recognize 0 Different promoter sequences What is the heat shock sigma 0 Sigma 32 What is the motility sigma 0 Sigma 28 What is the starvation sigma 0 Sigma 38 What controls transcription of the promoter for the gene encoding flagellin 0 Alternative sigma factor sigma D What is agellin used for 0 Protein used to build agellar filament What happens if sigD is not transcribed 0 Sigma D protein is not made What happens of sigma D protein is not made 0 The flagellin gene is not transcribed How was the activity of the flagellin promoter measured 0 Fluorescence microscopy Define a transcriptional fusion 0 Fusion of a promoter of interest to a reporter gene Define a reporter gene 0 Easily detected gene not normally present in he research system If P agellingfp is transcribed what is the conclusion 0 The cell was ON for transcription cell was green If P agellingfp was not transcribed what is the conclusion 0 The cell was OFF for transcription cell was not green Why is sigma D transcribed infrequently 0 Because It was at the end of the long operon I How do you know 0 It was experimentally moved to different positions closer to promoter which caused P agellin to be ON for transcription more frequently LECTURE NINETEEN TRANSCRIPTIONAL REGULATION IN PROKARYOTES II LECTURE TWENTY EXAMPELS OF PROKARYOTIC GENE EXPRESSION When RNAp binds to promoters wealltly it does what 0 Initiates basal low levels of transcription When a repressor binds to an operator sequence 0 RN Ap is blocked from binding to the promoter thus inhibiting transcription of a gene When an activator binds to a sequence away from the promoter 0 RN Ap is recruited to the promoter thus increasing levels of gene transcription Where do repressors bind 0 Operator sequences Where do activators bind 0 Sequences away from the promoter What are the genes under lac operon control 0 lacZ lacY and lacA Which proteins are made after lacllt operon transcription 0 LacZ LacY and LacA What does the lac operon do 0 Transcribed genes into proteins dedicated to breaking lactose into glucose What does LacY do 0 Transport lactose into the cell What does LacZ do 0 Cleaves galactose and glucose Is the promoter for the lac operon weak or strong 0 Weak I Why 0 It is far from consensus If only lactose is present the cell needs 0 Lac operon to be expressed to convert lactose to glucose When are lac genes turned on 0 In the presence of lactose What turns on the lac operon 0 CAP binding to the CAP site Is CAP an activator or repressor 0 Activator I Where does it bind 0 It is an activator so it bids to a sequence away from the promoter CAP site not to the operator sequence like a repressor When CAP binds to the CAP site what happens 0 RN Ap is recruited to the promoter I activating high levels of transcription When glucose is present what happens to the lac operon 0 Lac genes are turned off What is the repressor of the lac operon 0 Glucose Where does glucose bind 0 To the operator preventing transcription Define quorum sensing 0 A mode which bacteria uses to communicate with each other by emitting detecting and responding to chemical signals Using qurom sensing cells sense their own population density 0 Indirectly Cells directly sense 0 Autoinducer an extracellular signaling molecule Define autoinducer 0 An extracellular signaling molecule that diffuses out of the cell and into the environment What is LuxR 0 A transcriptional activator I What does it require 0 A high concentration of autoinducer to bind DNA How do LuxR and autoinducer activate transcription 0 As autoinducer extracellular levels accumulate it diffuses into the cell and binds to LuxR I LuxR autodinducer bind to DNA and activates transcription How does autoinducer accumulate 0 In proportion with cell density When does autoinducer concentration reach a threshold to activate gene expression 0 At the critical cell density Each species of bacteria makes a auto inducer 0 Structurally unjique Each species of bacteria can sense their own autoinducer 0 Sense Bacterium V fischeri regulates the expression of o Lucifierase Define lucieferase 0 An enzyme that emits light V fischeri provides for the squid 0 Counter illumination LECTURE TWENTYONE TRANSCRIPTION IN EUKARYOTES I Do eukaryotes have operons 0 NO Which RNA polymerase is used in eukaryotic transcription 0 Pol II Do eulltaryotes have sigma factors 0 No What happens to the mRN A transcript after eulltaryotic transcription 0 It is processed I How 0 5 capping splicing 3 polyadenylation What control transcription in eukaryotes o Promoter and regulatory sequences Define promoter 0 DNA sequence bound by general transcription factors which recruit RNA polymerase Define regulatory sequences 0 Define a core promoter 0 A set of sequence specific elements needed by RN Ap to initiate transcription I Where are elements located 0 Either upstream or downstream of 1 What do Pol I and Pol III transcribe o Specialized RNAencoding genes What does Pol II transcribe 0 Nearly all proteinencoding genes Describe transcription initiation 0 Binding of RNAp to a closed complex promoter 0 DNA strands separate to form an open complex 0 Initial transcription promoter escape What is the preinitiation complex 0 Pol II 6 general transcription factors and additional proteins bound at the promote What does the preinitiation complex do 0 Position Pol II over the transcriptional site and separate DNA strands at promoter Define TFIID 0 Transcription factor for RN Ap D that contains the TATA binding protein as a subunit What does TFIID do 0 It s subunit TBP binds to the TATA box in the core promoter What does TFIIA do 0 Helps bind TBP of TFIID to the TATA box What recruits TFIID 0 TFIIA What does TFIIB do 0 Binds to the TFIIB recognition element BRE in the core promoter What recruits RN Ap to TFIIF 0 TFIID TFIIA TFIIB What does TFIIF do 0 Stabilizes proteinprotein interactions What does TFIIE do 0 Recruit TFIIH What does TFIIH do 0 Mediates promoter melting by ATP hydrolysis and phosphorylated Pol II tail What is the order in which transcription factors join the preinitiation complex 0 TFIID TFIIA TFIIB TFIIF TFIIB TFIIH Define mediator complex 0 This interacts with the tail of Pol II and activators general transcription factors How does the preinitiation complex gain access to the DNA 0 Proteins are recruited to modify and remodel nucleosomes What are the steps of promoter escape 0 Promoter melting and phosphorylation of Pol II What happens between the steps of promoter escape 0 Abortive synthesis Describe promoter melting 0 TFIID holds the upstream promoter in place while TFIIH which binds downstream of RNAp uses ATP hydrolysis to feed dsDNA into RNA pol II thus causing strand separation Describe phosphorylation of Pol II 0 After Pol II is recruited to the promoter TFIIH phosphorylates the carboxylterminal domain Pol II tail to help Pol II escape general transcription factors and proceed to elongation Before elongation happens what occurs 0 Initiation factors and mediator complex are displaced Describe the chemical reaction of RNA synthesis 0 Beta and gamma phosphates are removed from the incoming ribonucleotide A phosphodiester bond forms between previously added NTP and incoming NTP Describe how nucleosomes are formed 0 H3H4 tetramer binds dsDNA and then two H2AH2B dimers join the complex What aids elongation in the presence of chromatin 0 FACT What is FACT 0 A heterodimer of two proteins SPT16 and SSRPl What does SPT16 of FACT do 0 Binds to the H2AH2B tetramer of a nucleosome What does SSRPl of FACT do 0 Binds to the H3H tetramer of a nucleosome How does Pol II continue elongation in the presence of chromatin 0 SPT 16 binds to the H2AH2B dimer ahead of Pol II and replaces the dimer as Pol II passes What helps in nucleosome assembly 0 SPT6 I Is this part of FACT 0 No When does capping occur 0 As the transcript emerges from Pol II Define capping 0 Adding of a methylated guanine 0 he 5 end of RNA What does RNA triphosphatase do 0 Removes a beta phosphate from the 5 end of Guanine In forming the 5 cap which phosphates are removed 0 Gamma phosphate from 5 end of RNA 0 Beta and gamma phosphates from guanine In forming the 5 cap what does guanyltransferase do 0 Transfers guanine to the 5 beta phosphate of RNA In forming the 5 cap what does methyltransferase do 0 It adds methyl groups to the newly added guanine What does the 5 cap do 0 Protects the end of RNA from degradation 0 Serves as a signal for the ribosome to bind and initiate translation LECTURE TWENTY TWO TRANSCRIPTION IN EUKARYOTES II What does the CTD tail do 0 Recruits polyadenylation enzymes What does transcription of polyA signal sequence do 0 Causes CstF and CPSF polyadenylation enzymes to transfer from CTD tail to RNA Define CstF 0 Cleavage stimulation factor Define CPSF 0 Cleavage and polyadenylation specificity factor When CstF binds to mRN A what happens 0 mRNA is cleaved into two pieces When CstF cleves mRNA into two pieces a short and a long piece what happens 0 Short piece is degraded and the long piece is further modified at the 3 end I What is another name for the long piece of RNA 0 Primary transcript When CPSF binds to mRN A at polyA signal what does it do 0 Recruits polyA polymerase PAP What does PAP do 0 Bind to the 3 end of mRN A and add the polyA tail What does the polyA tail do 0 Protect end of mRNA from degradation What do polyA biding proteins do 0 Bind too and stabilizes polyA tail What are the models for transcriptional termination 0 Torpedo and allosteric Describe torpedo model of termination 0 Rat 1 is loaded onto short mRN A and degrades RNA in a 5 I 3 direction until it reaches Pol II Pol II dissociates from DNA and transcription terminates I Why can t Rat 1 degrade primary transcript 0 Primary transcript is capped at 5 Describe allosteric model of termination 0 PolyA signal transcription leads to a decrease of pol II processivity causing pol II to spontaneously dissociate I Why is there a decrease of processivity I Conformation change in Pol II 0 Why I Proposed idea transferring of factors to mRN A tails might induce the conformation change LECTURE TWENTY THREE STUDY GUIDE Define primary structure 0 Amino acid sequence in a polypeptide chain Define secondary structure 0 Local structure arrangement of a short stretch of amino acids Define tertiary structure 0 Folded structure of polypeptide chain Define quaternary structure 0 Arrangement of multiple protein subunits in a larger complex Define domain 0 A part of protein structure independently folded and stable from the rest of the protein I What is it s level of structure 0 Tertiary What does Gal4 do 0 Activates transcription of GALl gene 100 fold Where does Gal4 bind o UASG Where is UASg 0 275 base paris away from Gall In the domain swap experiment what does it mean if Gal4 wild type bind DNA at UAS 0 Transcription of reporter gene is on In the domain swap experiment what does it mean if Gal4 mutant without an activating domain bind DNA at UAS 0 Transcription of reporter gene is OFF In the domain swap experiment what does it mean if Gal4 mutant without DNA biding domain bind DNA at UAS 0 DNA doesn t bind to UAS 0 Transcription of reporter gene is off Can DNA binding domains be swapped between eulltaryotes and prolltaryotes 0 YES Do DNA binding proteins from different organisms bind differently 0 No What classifies actuating domains 0 Amino acid content Each DNAbinding domain has what 0 Recognition helix Define recognition helix 0 One alpha helix inserted into the major groove Define homeodomain 0 A DNAbinding domain characterized by three alpha helices What happens to the three alpha helices of the homeodomain 0 Two form a helixturnhelix motif 0 The other is the recognition helix and inserts in the major groove Define zinc finger 0 A DNAbinding domain characterized by an alpha helix and beta sheet In zinc finger where is the zinc ion positioned 0 Between cysteine and histidine residues What does the zinc ion in the zinc finger do 0 Stabilizes domain folding and is important for DNA binding What do activators recruit 0 Transcriptional machinery and nucleosome modifiers What do nucleosome modifiers do 0 Add or remove chemical groups to histone tails to remodel the nucleosome thus helping increase expression of genes What do histone acetyltransferases do the chromatin 0 Loosens chromatin What do histone deacetylases do to the chromatin 0 Condense chromatin Describe how modification of histones gives accessibility of the promoter to transcriptional machinery 0 Activator binds enhancer which recruits HAT thus loosening the chromatin LECTURE TWENTY FOUR STUDY GUIDE What accounts for organismal diversity 0 Different genes and transcriptional regulators What do transcriptional regulators do 0 Bind to specific sequences regulatory binding sites on DNA to regulate transcription Define synergy 0 Interaction between two ore more objects to produce a combined effect greater than the sum of the individual effects Define cooperative binding 0 The binding of one protein to a DNA binding site which helps another protein bind to a nearby site Describe examples of cooperative binding of activators 0 A and B directly interacting with Protein X 0 A and B indirectly interacting with Protein X 0 Protein A recruiting nucleosome remodeler which uncovers the binding site for B 0 Protein A binding DNA associated with nucleosome which causes the DNA to unwind and expose binding site for B Where do repressors bind to repress transcription 0 DNA that is not overlapping the promoter Define competition 0 Repressor binds to a site that overlaps activator binding site Define inhibition 0 Repressor interacts with and blocks activator s activating domain Define direct repression 0 Repressor interacts with preinitiation complex leading to inhibition of transcription initiation Define indirect repression 0 Repressor recruits histone modifiers LECTURE THIRTY THIRTY THREE STUDY GUIDE is transcribed to 0 DNA mRNA is translated to a 0 mRNA protein What is the template for translation 0 mRNA In eulltaryotes where does transcription occurs 0 Nucleus In eulltaryotes where does translation occur 0 Cytoplasm In prolltaryotes where does transcription occur 0 Cytoplasm In eulltaryotes where does translation occur 0 Cytoplasm What is a codon 0 A set of three bases What is the first codon 0 AUG What are the stop codons 0 UGA UAA UAG What are features of an amino acid 0 Amino group carboxyl group proton R group How do you define a reading frame 0 Start and stop codon What does an open reading frame specify 0 A single protein How was the genetic code cracked 2 ways 0 1 Make transcripts consisting of one nucleotide 0 2 Make transcripts of alternating nucleotide What transcribes tRNA 0 Pol 111 What does transfer RNA encode 0 Noncoding RNA What is the structure of tRNA 0 Cloverleaf secondary structure What does the anticodon consist of what is made of 0 Three bases in tRN A In which orientation do codons and anticodon bind 0 Antiparallel What does the codon do the anticodon 0 Complementary base pair What is the wobble effect 0 A charged tRN A can interact with multiple codons What does the 5 nucleotide of the anticodon base pair with 0 3 nucleotide of the codon What is the alternative base 0 Inosine What is a silent mutation 0 A change in the basepair that still makes the desired amino acid What is a missense mutation 0 A change in the basepair that changes the codon to a different amino acid What is a nonsense mutation 0 A single base pair change to a premature stop codon What is a frameshift mutation 0 An addition of a basepair that leads to shift in the reading frame What is Google Genomics O In prokaryotes when does translation begin 0 While transcription is still occurring What is uncharged tRN A 0 tRN A without amino acid What is charged tRNA 0 tRN A with an amino acid attached to the acceptor stem How does aminoacyltRNA synthetase charge an amino acid 0 Attach amino acid to tRN A What is monocistronic 0 It means it has one open reading frame What is polycistronic 0 It means it has multiple reading frames 0 Happens when the genes are in operon What is the ribosome made of 0 Protein and rRNA What does an aminoacyltRNA synthetase 0 It charges a tRNA What are the sizes for large and small prolltaryotic subunits 0 30 and 50 2 70s ribosome What are the sizes for large and small eulltaryotic subunits 0 40 and 60 2 80s ribosome What is the large subunit also called 0 Peptidyl transferase center What does the peptidyl transferase center do 0 Form peptide bonds between amino acids What does the decoding center do 0 tRNA reads mRNA codons What is the small subunit called 0 Decoding enter What is the target of most antibiotics 0 Bacterial ribosome any thing with a 70s ribosome and 30s and 50s subunit Where does IF1 bind 0 Binds to the A site Where does IF3 bind 0 Binds to the E site Where does IF1 bind 0 Binds to the IF1 which binds to the A site What do IF2 and IF1 do 0 Will contact initiator tRN A and help it bind small subunit Where does the small subunit bind on the mRN A in prokaryotes 0 RES What is the RBS 0 A consensus sequence When the small subunit in prokaryotes binds RBS of rRN A what happens to the start codon 0 It is positioned in P site Once the small subunit in prokaryotes binds the RBS what happens with the start codon 0 Start codon is positioned in the P site When the start codon is positioned in the p site what happens to the initiator tRN A 0 It enters p site In eulltaryotes what blocks the A and E sites 0 eiF1 eiF1A and eiFS What is initiator tRN A charged with in eukaryotes 0 Met What does eiF4G do 0 Interact with polyA tail and polyA binding proteins I circular DNA structure How is mRN A recruited to small subunit 0 eIF4E binds to the 5 cap Small subunit and associated eIFs align initiator tRNA in P site by 0 Scanning mRNA 5 to 3 What begins termination 0 Stop codon in the A site Are there tRNAs that correspond to a stop codon 0 NO What does Class I release factors bind to the stop codon do 0 Release polypeptide chain from tRN A in P site In prokaryotes are all stop codons recognized by the same class I release factor 0 No In eukaryotes are all stop codons recognized by the same class I release factor 0 Yes What causes class I RFs to dissociate 0 Class II factor GDP exchange to GTP Explain the class I dissociation 0 Class II factor with GDP binds to A site GDP has high affinity to class factor 0 GDP is exchanged for GTP I kicks off class I 0 GTP has a high affinity for ribosome but it is hydrolyzed to GDP which kicks off GDP and class II factors After class II factors is kicked out of the A site are the B and P sites empty 0 No How do you empty the B and P sites 0 RF inserts in the A site 0 BFG kicks RRF into P site 0 Release of P and B site tRNAs What happens when tRNAs have been released from the P and B site 0 mRNA is released What stimulates ribosome dissociation 0 IF3 What is ribosome recycling 0 Removal of tRN As and mRNAs from ribosome which is then dissociated into large and small subunit What is the exon junction complex 0 Proteins Where are BICs located 0 Approximately 20 nucleotides upstream of BIC What do BICs serve as for the advancing ribosome 0 Guideposts If the ribosome comes into contact with an BIC what does it do 0 Keep translating How does translation terminate think of BIC 0 Stop codon with no BIC proteins downstream What is nonsensemediated mRN A decay 0 Degradation of mRNA with a premature stop codon What does the cell do if it reaches a stop codon without more BIC proteins ahead 0 Terminate translation What does the cell do if it reaches a stop codon and there are more BIC proteins ahead 0 Ribosome is displaced and proteins are recruited to degrade mRN A What do antibiotics target 0 Prokaryotes or both prokaryotes amp eukaryotes A lot of antibodies target what 0 Ribosomal and translational machinery What does puromycin do 0 Inserts within A site of ribosome If puromycin enters the ribosome where is the peptide bond formed 0 On puromycin If puromycin enters the ribosome where is the polypeptide chain added 0 On puromycin If the polypeptide chain is added to puromycin what happens to additional amino acids 0 They can t be added If the puromycin is in he ribosome what happens to the resulting protein 0 It is truncated and dissociates from the ribosome LECTURE THIRTY SEVEN STUDY GUIDE If an experiment procedure is performed in a controlled environment outside of a living organism 0 In vitro Is PCR in vitro or in vivo 0 In vitro What can t in vitro do 0 Replicate the precise cellular conditions of an organism Define in vivo 0 Performing an experiment or a procedure using a living organism Is EMSA Electrophoretic mobilityshiftassay in vitro or in vivo What does EMSA detect 0 ProteinDNA interactions Define DNA probe 0 A radioactively labeled dsDNA fragment generated containing a potential DNAbinding site How is radioactivity of DNA probe detected 0 Autoradiography In EMSA the protein of interest is purified and mixed with a probe in order to run 0 Gel electrophoresis Define free DNA 0 DNA fragment not bound to a DNA binding protein Define the left lane of EMSA 0 A control reaction in which DNA is not mixed with protein free DNA only Define the right lane of EMSA 0 Excess DNA mixes with protein 0 Does all of the DNA bind to protein I No I there is some free DNA available What migrates slower within the gel 0 ProteinDNA complex ProteinDNA complex migrating slower within the gel results in a 0 Shift in the location of radioactively labeled DNA How many bands would be seen if there was no proteinDNA interaction 0 One I What would it be corresponding to 0 Free DNA 0 Why I Protein is not radiolabeled Why do you use DNase footprinting 0 To identify location of proteinbinding site in DNA What are the two DNase footprinting assay reactions 0 Control and experiment I Describe them 0 Control no protein added 0 Experiment mix protein with probe What is DNase 0 An endonuclease that cleaves DNA phosphodiester bond Which DNA is not cleaved 0 DNA bound by binding protein I Why 0 DNA binding protein provides protection from cleavage Is gel electrophoresis performed on DNase footprinting assay results 0 Yes What is the left lane of DNase footprinting assay 0 Digested DNA fragments only separated based on size What is the right lane of DNase footprinting assay 0 Digested DNA fragments bound to DNA binding proteins based on size Define footprint 0 An absence of bands corresponding to site of protein bonding What is used to determine the DNA sequence of the footprint 0 Chainterminating gel sequencing gel What does a chainterminating nucleotide have vs normal nucleotide 0 3 H vs 3 OH When a normal nucleotide is at the end of the strand what happens 0 DNA synthesis proceeds When a chain terminating nucleotide is at the end of the strand what happens 0 DNA synthesis stops What are the reaction ingredients for a chainterminating method 0 Singlestranded DNA to be sequenced template strand primer DNA polymerase all four normal nucleotides one chainterminating nucleotide per reaction When running a chainterminating sequencing gel alongside DNase footprinting gel what can be determined 0 The exact sequence of the protected region I How 0 Read sequencing gel top to bottom within footprint to determine sequence LECTURE THIRTY EIGHT STUDY GUIDE Define ChIP chromatin immunoprecipitation 0 Used to detect genomewide proteinDNA interactions in living cells Describe ChIP 1 Begin with living cells 2 Add formaldehyde to cells 3 Lyse cells 4 Shear DNA 5 Immunoprecipitation Why do you add formaldehyde to cells 0 To chemically crosslinllt proteins to DNA Describe sonication 0 Process by which DNA is sheared into small fragments Describe immunoprecipitation in ChIP 0 Antibodies specific to protein of interest is attached in column I eluted out to get specific protein DNA 0 Proteins are removed from DNA through reverse crosslinllting 0 Remainder DNA is amplified and the sequence is used to identify specific regions Define molecular cloning 0 The ability to construct recombinant DNA molecules and maintain them in cells Define recombinant DNA 0 Artificial DNA that doesn t occur naturally Define restriction endonucleases 0 Enzymes that act like molecular scissors for DNA analysis Whether do restriction endonucleases cut 0 Digest DNA at specific sequences OOOOO What was the first restriction enzyme discovered EcoRI What does EcoRI recognize and cut 0 5 GAATC 3 What is produced after EcoRI recognizes and cuts GAATTC 0 A sticky end What does agarose gel electrophoresis use to separate segments 0 Size What does a replication origin ori allow for 0 Plasmid to replicate independently of chromosome What is the gene encoding resistance to antibiotics used as 0 A selectable marker to kill bacterial cells that fail to take up a plasmid Define a cut site 0 A site where a restriction endonuclease will cleave Define multiple cloning site 0 A cluster of restriction sites grouped together Define plasmid vector 0 Backbone for cloning Define insert 0 A fragment of DNA to be cloned into the vector typically a gene of interest or a promoter What is used to amplify insert 0 PCR The endonuclease digests what 0 Both the plasmid vector and insert I And produces what 0 Compatible sticky ends After both the plasmid vector and insert have been digested what happens 0 The insert is introduced to the plasmid vector Once the insert is introduced in the plasmid vector what happens 0 Sticky ends of insert and vector anneal and DNA ligase seals the nick How is the recombinant plasmid propagated 0 Transformation by competent E coli cells Once the plasmid is incorporated in the bacteria what happens 0 The plasmid has its own ori so it will replicate independently of bacterial chromosome as bacteria plate growth Problem you have a lot of e coli but not all e coli has the plasmid vector 0 Solution plate ecoli on agar plate with antibiotic so everything dies but the cells resistant to antibiotic with plasmid present 39 Only competent cells will survive
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