Biochemistry 465 Your Printed Name: Section I and onlyApril 15, 1999 Your SS#: Exam #2Prof. Jason D. Kahn Your Signature: You have 80 minutes for this exam.Exams written in pencil or erasable ink will not be re-graded under any circumstances.Explanations should be concise.You will not need a calculator for this exam, and no other study aids or materials are permitted.Useful equation:Θ = P / (P + K)BCHM465 Exam 2 4/15/9921 . (20 pts) Footprinting and protein-DNA bindingThe autoradiogram on the left below shows DNAse I footprinting of a sequence-specific DNAbinding protein to a labeled restriction fragment. The four lanes at the right show increasing totalprotein concentrations, with each lane having about 5 times as much DNA as the one to its left. Someof the data on the binding curve at the right came from the experiment on the left.(a; 6 pts) Indicate in the box in the center below where sites A and B are on the gel. Explain yourreasoning briefly:No DNAseNo ProteinIncreasing Protein123456Answerfor (a):⇓0.000.250.500.751.0010-10 M10-9 M10-8 M10-7 M10-6 M10-5 MBinding CurveSite ASite BFractional Saturation (Θ)Total [Protein]Note that the gel lanes do not necessarily correspond to particular points on thecurves above.(b; 4 pts) Using the binding curve above, determine numerical values for the protein’s dissociationconstants (Kd’s) from sites A and B.BCHM465 Exam 2 4/15/993(c; 4 pts) How would the curves above change (qualitatively: which direction would they shift?) ifthe experiment were done at higher salt concentration? Why?(d; 6 pts) The gel mobility shift or EMSA experiment is often used very qualitatively to identify thepresence of a low-abundance sequence-specific DNA binding protein in a complicated mixture like anuclear extract. Suggest why the EMSA is a better assay for this purpose than a DNAse I footprint(hint: there’s very little of the protein of interest in the mix). Why is it often necessary to include asource of non-specific DNA (e.g. poly d[A-T]) in the reaction mix?BCHM465 Exam 2 4/15/9942. (20 pts) Specificity and Structure(a; 2 pts) What is the single most common general structural motif for a protein-DNA interaction?(b; 3 pts) Why do sequence-specific RNA binding proteins never recognize fully double-strandedRNA?(c; 5 pts) Draw a hydrogen bonding interaction between the glutamine side chain and a nucleic acidbase to which it can make two hydrogen bonds when the base is in a Watson-Crick duplex.+H3NNH2CO2-OBCHM465 Exam 2 4/15/995(d; 5 pts) Why are zinc fingers especially suitable for designing artificial DNA binding domains withuser-defined specificity? Why are they always found to be at least dimeric if not multimeric DBD’s?(The answers are related).(e; 5 pts) What do we mean by the hydrogen bonding matrix that a protein “sees” upon approachingDNA? Give a specific example of how this allows discrimination between two different base pairs.BCHM465 Exam 2 4/15/9963 . (20 pts) DNA replication(a; 8 pts, 2 each) In the fundamental mechanism for nucleotide incorporation by DNA polymerases,what reaction provides proofreading (removal of a misincorporated base)? Why is it important forthis mechanism that extension using the 3′ end of a misincorporated bases is slow? It what way doesthis process spend energy? Why is coupling error correction to an irreversible process essential?(b; 5 pts) What are the important players in the active site of T7 DNA polymerase and many otherphosphoryl transfer enzymes? What do they do (how do they activate the reaction)?BCHM465 Exam 2 4/15/997(c; 4 pts) Sketch a replicating bacterial chromosome theta structure as seen during exponentialgrowth, assuming a 20 min doubling time and 40 min required for chromosome replication. (Anytime point during the cell cycle will do.)(d; 3 pts) Why is the replicative DNA polymerase III a dimer?BCHM465 Exam 2 4/15/9984. (20 pts) Transcription(a; 3 pts) Phage T7 RNAP is a single 100 kD protein that synthesizes RNA quite well. E. coli RNAPhas at least four different subunits and a mass of about 450 kD. Eukaryotic RNAPs ~2000 kD.What’s all the rest of the mass of cellular RNAPs for, if not chemistry?(b; 6 pts) Sketch the nucleic acid components of the transcription complex during elongation. Whydo RNA polymerases absolutely have to be processive? What’s the opposite of processive?(c; 6 pts) How could we “walk” an RNA polymerase ternary complex stalled at position +19 on thetemplate sequence below to position 23? What is the advantage in having a His6 tag or other affinitylabel on the enzyme if we want to walkit further, e.g. specifically to position25 and not further?...ATGATGAAGGTGGAGATGACAAGTCGATCGAAAA......TACTACTTCCACCTCTACTGTTCAGCTAGCTTTT...+15′ AUGAUGAAGGUGGAGAUGARNA ALREADY MADE:RNAP+19+23 +25BCHM465 Exam 2 4/15/999(d; 5 pts) Phage T4 encodes its own sigma factor and inactivates the host sigma factor. What effectwould this have on cellular transcription, and what advantages does it confer for the phage?5. (20 pts) Miscellaneous: Chromatin, telomeres, genomics(a; 4 pts) The measured ∆Lk induced by RNA polymerase binding is about -1.7 (18 bp), and yet itseems to unwind only about 12 bp. A nucleosome wraps about 1.7 turns of DNA around itself butinduces a ∆Lk of only -1. Give brief possible explanations for these two puzzles.(b; 6 pts) Give a brief sketch and explanation of how shotgun sequencing of genomes works.BCHM465 Exam 2 4/15/99 10(c; 7 pts) Why do organisms with linear chromosomes (like us) need telomerase? What doestelomerase use as a template? Why is telomerase under study as an anti-cancer target?(d; 2 pts) When we speak of a “rotationally positioned nucleosome” what is or is not rotating?(e; 1 pt) What creature from science fiction of the late 1960’s is brought to mind by bacterial DNAreplication?Do Not Write Below This LineScore: Question 1: out of 20: Footprinting and protein-DNA bindingQuestion 2: out of 20: Specificity and StructureQuestion 3: out of 20: DNA replicationQuestion 4: out of 20: TranscriptionQuestion 5: out of 20: Miscellaneous: Chromatin, telomeres, genomicsTotal: out of