Biochemistry 465 Your Printed Name: Section I and onlyMay 21, 1999 Your SS#: Final ExamProf. Jason D. Kahn Your Signature: You have 120 minutes for this exam, which is worth 200 points. Thus you get more “points per minute”than for the midterm exams.Though each question has several parts, all of the parts are completely independent of each other. Inother words, don’t give up if you can’t answer part (a).Explanations should be concise.You will not need a calculator for this exam, and no other study aids or materials are permitted.There will be a viewing on Monday, May 24, from 3-4 p.m., in Chemistry 2507 (next tomy office in the Biochemistry wing).Final grades will be available only through MARS or at the viewing.BCHM465 Final Exam 5/21/9921 . (40 pts) DNA Repair and Connections to other Processes(a; 10 pts) Which DNA repair system repairs bulky adducts like BPDE bound to one strand of DNA?In E. coli, what proteins comprise the repair system in question? What makes DNA repair unusual inbiochemistry in terms of enzyme specificity, relative to (for example) enzymes involved inintermediary metabolism?(b; 12 pts) In prokaryotes, the template strand of actively transcribed genes is repaired moreefficiently than DNA elsewhere in the genome. Why does this make biological sense ? In general,what is the key operation that the cell generally does not want to do with damaged DNA ?Transcription-repair coupling in eukaryotes is conceptually similar, with one important difference inregards to what happens to the RNAP. What is this difference, and why does it make sense?BCHM465 Final Exam 5/21/993(c; 8 pts) Sketch the pathway for base excision repair or methyl-directed mismatch repair or transcription-coupled repair in prokaryotes, whichever one is your favorite.(d; 4 pts) Repair of inter-strand crosslinks in DNA requires recombination. Why (in principle, noneed to draw a pathway)?(e; 6 pts) Briefly, how does p53 act as a tumor suppressor ?BCHM465 Final Exam 5/21/9942. (40 pts) Translation(a; 11 pts) Draw the chemical steps in charging a tRNA with an amino acid. What class of enzymes perform this reaction? In what sense do they determine the fidelity of translation ?(b; 6 pts) What are the names and functions of the two ribosomal subunits in E. coli?(c; 3 pts) Why is translation called translation ? In other words, what is the connection between thevernacular and scientific uses of the word “translation?”BCHM465 Final Exam 5/21/995(d; 11 pts) What is the function of EF-Tu in translation? Sketch the reaction cycle involving EF-Tu .How does its GTPase activity provide kinetic proofreading ?(e; 9 pts) Give some of the evidence for the critical role of the 23S RNA as the catalyst for peptidyltransfer, as opposed to its being simply a scaffolding element.BCHM465 Final Exam 5/21/9963 . (35 pts) DNA Recombination and Evolution(a; 15 pts) The sketch below shows the initial configuration for site-specific recombination betweenthe AttB and AttP sites in a plasmid model system for lambda phage integration. Label thesuperhelical nodes with their signs. Draw the product of the recombination reaction catalyzed by IHFand Int, with its superhelical nodes. What is the linking number change in the reaction? What doesfact that there is a definite answer to this question tell us about the mechanism of site-specificrecombination , and why does this make sense in terms of evolution of mobile DNA?↓↓↓↓BCHM465 Final Exam 5/21/997(b; 10 pts) Give two evolutionary rationales for the benefits of recombination. Why do transposableelements persist in our genomes (what phrase did we co-opt to describe them)?(c; 10 pts) Sketch the Holliday junction (in the crossed over representation) which would eventuallyresult from invasion of the RecA coated single strand as indicated below. Sketch the product ofbranch migration by the RuvAB motor, and draw/explain how resolution by RuvC always results ina heteroduplex segment but may or may not exchange flanking genetic markers .→→→→BCHM465 Final Exam 5/21/9984. (40 pts) RNA Splicing and Chemistry(a; 9 pts) We discussed the idea of paradigm shifts in science. How did the discovery of self-splicingof group I introns change a reigning paradigm in biochemistry, and how did this affect ideas on theorigin of life (what makes RNA special)?(b; 9 pts) The sketch below shows the group I intron after the first step of splicing. Redraw themolecule in the space to the right to prepare it for the second step, indicate the transesterification ofthe second step on your diagram, and show the products below .BCHM465 Final Exam 5/21/999(c; 6 pts) The spliceosome, which helps process pre-mRNA, is about the size of a ribosome anddoes simpler chemistry. Give two reasons that the ribosome is much better understood .(d;16 pts) You are given (1) the DNA oligonucleotide on the left below. (2) a solid support whichbinds biotin very tightly, (3) in vitro transcription, (4) reverse transcription and PCR, and (5)RNAse H. Sketch a selection-amplification scheme for isolating RNA sequences which have RNAligase activity, from the pool of semi-random RNA on the right. Biotin5′ GCGTAGCTCAG5′ GGUGGAAACGCAUCGAGUCCCACrandom partconstant 3′ regionpppBCHM465 Final Exam 5/21/99 105. (45 pts) Regulation of Transcription and Pedagogy(a; 12 pts) A genetic screen for E. coli unable to grow on lactose turned up a mutant lac repressorwhich cannot bind IPTG or other inducers but is otherwise functional. How could you test to makesure that the mutation was in lac repressor and not, for example, in β-galactosidase? It was foundthat the mutant phenotype could be suppressed by a change in the promoter region. What might thischange be ? What effect would the suppressor mutation have on lac operon expression in the absenceof lactose or glucose, in an otherwise wild-type cell?(b; 7 pts) What are the functions of the eukaryotic general transcription factor TFIID , which includesthe TATA box binding protein? What protein provides a roughly analogous activity in prokaryotes (the protein in question has other activities as well)?(c; 6 pts) Histone acetylases and deacetylases are important in eukaryotic