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WWU CHEM 473 - CHEM 473 Exam 2

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Chem Biol 473 Exam 2 May 11, 2011 Name___________________Please make your answers CONCISE and LEGIBLE!!!!!!!!1. (4 points) Why would ribosomal backtracking not work as a proofreading system for protein synthesis in the same manner that backtracking works for transcription?Backtracking would cause complete loss of the nascent peptide from the tRNA in the P site.2. (10 points) What is the purpose of the HIV protease?It cleaves the HIV polyprotein.This protease is an aspartate protease, meaning that it has 2 asp residues that are required for activity. These residues have different pKa values, of 3.5 and 5.3. The pH optimum for theenzyme is 4.4. What does this tell you about the reaction mechanism? What would be the %activity of this enzyme at pH 6? (This is a calculation.)Mechanism is by GAB catalysis—one asp must be protonated, the other deprotonated.At pH 6, the deprotonated asp is still deprotonated. How much of the protonated asp remains?6 = 5.3 + log A/HA5 = A/HAXHA = 1/6 = 17%3. (4 points) In the genome of E.coli, for example, there are 7 copies of the operon that produces rRNA, but only single copies of the genes that code for ribosomal proteins. Explain this discrepancy.rRNAs are required in stoichiometric amounts, whereas mRNAs for ribosomal proteins can be translated multiple times.4. (10 points) Knowing the mechanism for the production of ile-tRNA by its cognate synthetase, predict whether 32P-labeled ATP will be produced when each of the following sets of components is mixed with the synthetase. Explain your answers.a. ATP and 32PPi. no label—first step is formation of amino acyl adenylate.b. tRNA, ATP and 32PPi. Again, no label.c. Isoleucine, ATP and 32PPi. Labelling will occur as this step in the mechanism is reversible. ATP + leu ↔ AMP-leu + PPi5. (5 points) Think about the mechanism for peptide bond formation during translation. Explain how formylation of the initial methionine in bacteria might make sense in terms of this mechanism.Formylation blocks the N-terminus of the initiator met ensuring that its α-carboxyl group isthe one free to bond with the next aa.Formylation also is recognized by IF2 and therefore is required for initiation of translation.6. (10 points) In the paper, Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 controls HIV-1 in vivo, the authors used two different types of nuclease molecules in order to perform and verify the results of their experiments. Please identify these nucleases and explain how and why they were both used for this study. Be sure to include a diagram in your answer!ZFN targets endonulease cleavage of CCR5Cel-1 targets disrupted DNA double helices that result from non-homologous end joining following ZFN cleavage.7. (7 points) A student in your lab conducts a genetic enrichment/selection study on a DNA-binding protein to determine its specificity and consensus binding sequence. The base pair probability data from 230 independent sequence reactions is shown below.Based on this data, answer the following questions:a. How long is the family of sites recognized by this protein? What is the consensus sequence? Can you determine what type of DNA binding protein this is based on this sequence?6 bp: 5’T-A/G-A/C-T/G-T/C-A This is a palindrome so the DNA BP is at least dimeric.b. In the first draft of the paper discussing this experiment, your student writes that “based on the recovery data, the protein appears to make contacts with a run of six base pairs in its DNAtarget”. Is this conclusion correct based on the data shown? Why or why not?This is not necessarily correct. The data indicate where contacts for specific recognition are. Other non0-specific contacts likely occur with additional base pairs outside the recognition domainThat the protein displays 2-fold symmetry and is probably a


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WWU CHEM 473 - CHEM 473 Exam 2

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