Exam 3 Study Guide
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Popular in Biochemistry
This 11 page Study Guide was uploaded by Luke Holden on Sunday March 20, 2016. The Study Guide belongs to 3050 at Clemson University taught by Dr. Srikripa Chandrasekaran in Winter 2016. Since its upload, it has received 82 views. For similar materials see Essential Elements of Biochemistry in Biochemistry at Clemson University.
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Date Created: 03/20/16
BCHM EXAM 3 Study Guide Understand onegene oneenzyme hypothesis The hypothesis linked DNA to protein because every protein/enzyme needs one gene (DNA that codes for a certain message). Gene and protein have an exclusive relationship An example of this is for the synthesis of Arginine. This comes from a gene called arg. 1 that will make enzyme 1 and convert precursor to Ornithine which makes gene called arg.2 that exclusively makes enzyme 2 that converts to Citrulline which makes gene arg. 3 that makes enzyme 3 and converts to Arg. (see picture below) Will it With extra With extra With extra With extra Grow? Precursor Orinithine Citrulline Arginine Wild Type Yes Yes Yes Yes Yes Arg, 1 No No Yes Yes Yes *mutation Arg. 2 No No No Yes Yes mutation Arg. 3 No No No No Yes mutation * If you see: Δ this means a mutation Understand significance of template and sense strand of DNA The template strand is the strand of DNA that is transcribed and the sense strand of DNA is the nontemplate strand. Since the mRNA is transcribed in the 5’ to 3’ direction, the sense strand and the mRNA strand are identical except where there would be a T on the sense strand there is now a U on the mRNA strand. Differentiate between pro and eukaryotic transcription Prokaryotes promoter at 35 & 10 bp, Rho factor termination, lac and trp operon Eukaryotes TATA (25 bp) CAAT (50) GC (80) box, Enhancers/silencers that modify gene expression (thousands of bp downstream binds to an activator, loops entire DNA and activator starts interacting with RNA polymerase and starts transcription),3 different types of RNA polymerases, mRNA processing (5’ capping, 3’ poly A tail, splicing to remove introns ) 1. Promoter sequences and interaction with RNA polymerases A bunch of transcription factors (TF) bind to the TATA box (eukaryotic promoter sequence) and attract RNA polymerase 2 (has to be phosphorylated) (eukaryotes) Initiation, Elongation and Termination of transcription in pro v/s eukaryotes Transcription: o RNA polymerase choose a template strand (can pick either one) o Terms Non template strand (sense) RNA polymerase goes 5’3’! o Background information Promoter region of nucleotides 20200 bases upstream of the open reading frame Open reading Frame (ORF) This is the gene of interest that will be transcribed Promoter 10 bp before ORF in prokaryotes Promoter 35 bp before ORF in eukaryotes o When she gives us a DNA strand or mRNA strand she will always tell us which is which o Cheat code: THE NONTEMPLATE STRAND IS THE SAME THING AS THE mRNA STRAND (EXCEPT “T” IS EXCCHANGED WITH“U”) RNA Polymerase The enzyme that reads DNA to synthesize RNA Gene specific Subunit Structure o Five poly peptides o Core enzyme: ααββ’(i) o o Sigma factor binds to the promoter and prevents DNA from going back into heterochromatin Promoter recognition Binds to the core enzyme o Function of the subunits: α promoter binding and assembly and regulation β’ DNA Binding β Catalytic site (polymerization part) (The part that RNA is no for (i) structural role , restores activity Sample Question: I will compare polymerases (DNA + RNA) Prokaryotic Transcription o 3 Stages Initiation and Elongation RNA Poly binds to initiation After 10bps = Elongation Elongation is the process of transcribing mRNA. Termination Rho (ρ) factor ATP dependent helicase that stops transcription in prokaryotes o ρ Attaches to mRNA and follows behind the RNA poly o When RNA poly 3 stalls, the ρ factor can catch up and essentially knock of the mRNA strand o This is kind of like you are swimming a way from a shark and then catch a cramp…. You die Eukaryotic Transcription RNA Polymerase Location Function I Nucleolus Transcribes large rRNA’s II Nucleus Transcribes mRNA and snRNA’s III Nucleus Transcribes tRNA 5s rRNAs EUKARYOTIC POLYMERASE CANT INITIATE TRANSCRIPTION LIKE PROKAROYTES!!!!! o Initiation DON’T HAVE TO KNOW SPECIFIC TRNASCRIPTION FACTORS Several transcription factors TF’s bind to TATA box Polymerase binds to the promoter TFIH gets polymerase going via phosphorylation Promoters: Promoter Consensus Position Function Sequence TATA TATAAAA 25 Indicates transcription Start Site CAAT GGCCAATCT 50 Indicates Strong promoter (High Rate) GC Box GGGCGG 80 Indicates House Keeping gene (All the Time Enhancers Assist in the formation of the transcription complex 10002000 bp away DNA will bend back on itself and attach IF to promoter Gives the polymerase a sort of shove Compare and Contrast Prokaryotic and Eukaryotic Characteristic Prokaryotes Eukaryotic Polymerase RNA Poly: Can do it all! RNA poly I: large RNA rRNA RNA poly II: mRNA mRNA RNA poly III: tRNA small tRNA tRNA Initiation Factors σ factor further binds to theTF IIs bind to the promoter promoter o Termination RNA poly reaches the end of the terminator regions of DNA Terminator: Poly A consensus sequences that code for hairpin structures The hair pin forms kind of like Velcro where in latches on to itself and then it falls off. mRNA splicing and significance mRNA splicing takes place only in eukaryotes after termination. It is in the removal of introns and the ligation of exons. This process occurs inside the nucleus and adds genetic variation due to splicing at different locations. What is the structure of a transcription factor? Know properties of the Transactivation domain and DNA binding domain. Know examples and salient features of some of the common domains in transcription factors. o DNA Binding Domain Rich in α helices Interact with the major groove with h bonds Salt bridges and hydrophobic interactions Can bind to promoters and enhances o Active domain Mainly acidic amino acids Bind to other factors, RNA poly II and activators How is the lac operon regulated in prokaryotes? Know how regulation is controlled in the presence of various metabolites. Prokaryotes prefer to use Glucose for energy but when glucose isn’t present, the prokaryote will settle for making lactose to break down into glucose. When Glucose is high in the prokaryote, the lac operon is therefore off and when glucose is low, the lac operon is turned on. +Glu +Glu Glu Glu +Lac Lac +Lac Lac Transcription + Lac operon and use Lac operon +++ Lac /+ try to turn it on level some of lactose but completely operon b/c no Glu but not a lot b/c glucose is off “pumped up” quickly turns off present (1x) (50x) b/c no Lac Repressor Bound to allolactose Bound to Bound to Bound to operator operator allolactose CAPcAMP No CAPcAMP No CAP Increased Increased CAP cAMP CAPcAMP cAMP + Lactose Lactose Transcription No transcription Derepresesed Repressed Repressor is not bound Repressor is bound to the operator + Glucose Glucose No cAMP= no cap Increase in adenylation No activation Increases cAMP= Increase in CAP What is the difference between lac and trp operons? Operate inversely from each other Trp operon is on when trp is absent from the prokaryote, repressor bound when there is plenty of trp present Lac operon is on when lac is present in the prokaryote, repressor bound when lactose is absent Know the different modes of transcriptional regulation in eukaryotes (histone modification, epigenetic regulation, regulation by activators, repressors, transcription factors and mediators) Epigenetic Regulation (DNA modification) o Addition of methyl groups to cytosine (methylated) Happens in CpG islands It is associated with reduced transcription Methylated DNA can change the way the DNA is read Can be due to diet and stress o Histone modification add acetyl to lysine or arginine loosens DNA and enhances DNA Enhancers activators and Mediators o Activator binds to enhancer and stimulates transcription o Mediator proteins mediate interaction between enhancers and DNA o Transcription initiation complex: TF, Activators, RNA poly II and mediator o LCR Locus Control Region Example of enhancer region in DNA Depressors and Silencers o Repressors bind to silencers o Inhibits DNA o You need to know this picture o Describe the synthesis (and specificity) of aminoacyltRNA’s. (tRNA Charging) Synthesis: o two step reaction: First an AA is attached to a phosphate group forming an anhydride bond by using 2 ATP Then, the AA is then attached to the tRNA by replacing the phosphate with the tRNA as shown below: o Specificity: one or more synthases per AA tRNA/ anticodon recognition elements Some even have proofreading ability How is posttranscriptional regulation of mRNA maintained in eukaryotes? What is alternate splicing and how do miRNAs and siRNAs function? Post transcriptional o Removal of introns o Splicesome Enzyme complex snRNP and snRNA’s (RNA interference) can cut it in different ways MicroRNA’s and siRNA’s small repeats that bind to dicer by forming a hairpin and then degraded or block translation Steps of RNA interference The miRNA folds on itself Dicer comes and makes it into smaller fragments miRNA fragments come and bind to the protein complex the protein complex and miRNA binds to the RNA Then it is either degraded (miRNA) or blocks transcription (siRNA) Summarize the main features or characteristics of the genetic code, including start & stop codons. Codon A threebase sequence of mRNA 1. Sequence codes for a specific amino acid 2. Some amino acids can be formed by more than one codon Start codon AUG 1. Point where the genetic code is first read Stop codon 1. Point where the genetic code is finished being read Anticodon Three base sequence that pairs with codon on the tRNA 1. Both sequences are given in 5’ to 3’ direction. 2. Example: Codon UGC binds to anticodon GCA What is meant by base pair “wobble” and why might it be beneficial? On the third position of the codon (the 3’ position on the codon and the 5’ position on the anticodon), that bp can be a different nucleotide match as compared to the other ones. This allows for the translation mutation s such as a nonsense or a silent mutations to be avoided. This is because even if the codon is read incorrectly, the protein will be the same. Name & describe the details in each of the three stages of protein synthesis, including the structure & functions of ribosomes and any factors involved. Prokaryotic translation: o Initiation: This is the first step when the mRNA strand arrives at the ribosome Formation of the initiation complex The shinedalgarno sequence: this is a purine rich sequence upstream from the AUG codon. Recognition of this sequence is crucial and is recognized by the 16s rRNA strand. 1. Consensus sequence: AGGAGG Sequence of events: 1. IF1 and IF 3 bind to the 30 s subunit IF1 blocks the A site until the first tRNA is in place IF3 blocks the 50s subunit from binding the 30s subunit. 2. 30s subunit binds to the shine dalgarno sequence 3. 30s subunit slides in place over the AUG start codon (5’3’) 4. Insure that the first AUG start codon is located 5. IF2 with GTP binds to the 30s Asite 6. IF1 is then displaced and the fMettRNA (ONLY PROK) (Insert Picture) 7. GTP is hydrolyzed which makes IF2 and 3 fall off 8. The 50s subunit then comes and binds the complex thus creating the characteristic ribosome o Elongation: The EfTu protein carries the charged tRNAs and then hyrolzes GTP to attach it to the codon in the A site. GTP is then rehposphrylated and the EFtu is then reattached with a charged tRNA. peptidyl transferase (in the large ribosomal subunit)then attahces the growing polypeptide chain to the charged tRNA in the A site. EFGGTP moves the ribosome in the 3’ direction which shifts the tRNA’s over a site. The energy released from the GTP causes the ribosome to change conformation. o Termination: The stop codon calls for the initiation factor which comes and bids to the A site This converts peptidyl transferase into hydrolyase which cuts the polypeptide away from the tRNA and Ribosome falls away o Multiple ribosomes bind to the same sequence of mRNA and forming polysomes which amplify the protein. Describe & exemplify how some antibiotics function by impairing transcription and/or translation. Prokaryotes only: o Tetracycline: blocks the aminoacyl tRNA from binding to the a site o Chloamphenicol: blocks the peptidyl transferase on the ribosomes o Erythromycin: blocks the translocation of the ribosomes o Cordycepin cordycepin is similar to adenosine, some enzymes cannot discriminate between the two. Therefore, it can participate in certain biochemical reactions (for example, be incorporated into an RNA molecule, thus causing the premature termination of its synthesis). Eukaryotes only o α amanitin blocks transcription of mRNA by binding to RNA Poly II ON HER POWERPOINT (LECTURE 13) WE ONLY HAVE TO KNOW THE ONES THAT SHE BOXED! Identify the different type of mutations (missense, nonsense, silent, insertion and deletion) Translation mutations: o Missense: This is where an incorrect Amino acid is inserted into the protein sequence. This can be detrimental as it can influence misfolding of the protein and thus altering its function o Nonsense: This is a mutation where a stop codon is sequenced and causes early termination of translation. Usually results in dysfunctional protein that is useless o Silent: This typically occurs during the wobble hypothesis where the third nucleotide of the codon is switched for another nucleotide. However, to our luck, they code for the same protein and thus the protein goes on about its life. You would not know these occur unless you looked at the genetic code. Transcription mutations: o Frame shift mutations: can be an insertion or a deletion that cause the frame of reading (codon) to be shifted over or back one Insertion: This is when a n extra nucleotide is inserted into the mRNa strand that cause the frame to be moved over in the 3’ direction. Deletion: This is when a nucleotide is deleted and the frame is shifted over to the 5’ direction Given either sense, antisense or the mRNA strand, identify the type of point mutation shown AntiSense strand: 5’ ATGCCGATTACGGATTCCGGAAT mRNA Stra: 3’ UACGGCUAAUCCCCUAAGGCCUUA o An insertion occurred Know the difference between transition and transversion mutation Transition is when a bp change occurs where a purine is switched for a purine and a pyrimidine is switched for a pyrimidine Transversion This is a bp change that occurs where a purine is switched for a pyrimidine
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