BIOL0280 Third Midterm Examination 2014

Indicate which statement is true of tetrapyrrole biosynthesis in both animals and plants: A) δ -aminolevulinate is a tetrapyrrole precursor. B) Carbon atoms of tetrapyrroles are derived from glycine and succinyl-CoA C) Carbon atoms of tetrapyrroles are derived from glutamate. D) A total of 4 δ-aminolevulinate molecules are used to synthesize 1 heme molecule. Circle the correct answer. 2. [4 points] Draw the structure of adenosine in the space provided below. Your answer must be completely correct to receive credit. H 4 points for completely correct 3. [4 points] Describe the regulation of ribonucleotide reductase at the primary regulatory sites. Please include in your description all the molecules that regulate these sites and for each regulator, the rationale behind the regulation and whether the regulation is positive or negative. The primary regulatory sites that effect the overall activity of the enzyme and NOT substrate specificity(-2 if answer describes primary regulation as effecting specificity), are positively regulated by ATP (1 point) which is an indicator of energy charge (1 point) and negatively by dATP (1 point) which is a feedback regulator (1 point).

Which of the following compounds directly provide atoms to form the purine ring? A) Aspartate B) Carbamoyl phosphate C) Glutamine D) Histidine E) Tryptophan F) Lysine G) Glycine H) Ornithine Circle all correct answer(s) More than one answer may be correct.2 points for all correct, 0.5 point off for any incorrect or 0.5 point off for any missing answer. 0 points for circling all answers. 5. [4 points] Draw the structure of guanine and cytosine correctly indicating the hydrogen bonding between them in the space provided below. Your answer must be completely correct to receive credit. 4 points for completely correct 6. [4 points] Explain how DNA binding proteins can discriminate between AT and TA base pairs. Make sure to describe exactly where on the DNA proteins would bind and the types of bonds formed between DNA and proteins and how these bonds provide the specificity to discriminate AT from TA base pairs. Proteins make hydrogen bonds to nucleotide bases (1 point) in the major groove of DNA (1 point). The arrangement of hydrogen bond acceptors and donors differs in AT and TA base pairs, allowing proteins to differentiate the two possibilities (2 points).

Which statement about the carbamoyl phosphate synthetase of mammals, which is used for pyrmidine biosynthesis, is true? A) It is located in the mitochondria. B) It is located in the nucleus. C) It uses NH4+ as a nitrogen source D) It uses glutamine as a nitrogen source Circle the correct answer 8. [2 points] Which of the following statements about topoisomerases is incorrect? A) Type II topoisomerases change the linking number in increments of 2. B) They break and reseal phosphodiester bonds. C) Type I topoisomerases can introduce positive supercoils. D) Type I topoisomerases form covalent intermediates with their DNA substrates. E) Type II topoisomerases can use ATP hydrolysis to form negatively supercoiled DNA from relaxed DNA in E. coli. Circle the correct answer. 9. [4 points] The DNA of virtually every cell is _under_____-wound (under or over). In eukaryotic cells, DNA is wrapped around __histone_________ proteins to form a _____nucleosome__ core particle that can be compacted into a __30 nm__________ fiber. Please fill in the blanks above with the correct terms. 10. [5 points] For the following statements about DNA replication in E. coli, indicate whether the statement is true or false (by circling T or F). If the statement is false, provide a brief correction on the adjacent line. T or F It occurs at a replication fork _______________________________________________ T or F It starts at a unique locus on the chromosome ___________________________________ T or F It proceeds with a single replication fork_____two replication forks_______________ T or F It uses RNA transiently as a template strand___RNA is used as a primer___ T or F It involves discontinuous synthesis on the leading strand ____discontinuous on lagging strand_ 11. [2 points] The enzyme which removes the RNA primer from the Okazaki fragment is A) Deprimase B) RNAse H C) DNA polymerase I D) DNA polymerase II E) DNA polymerase III Circle the correct answer.

Which is not a property of E. coli DNA polymerase III? A) It is responsible for incorporating most of the nucleotides in the lagging strand. B) It completes synthesis of the entire leading strand prior to aiding in the synthesis of the lagging strand. C) It contains a 3′ to 5′ exonuclease activity. D) It is a large protein complex containing more than five subunits. E) It contains a DNA helicase activity for unwinding DNA. Circle the correct answer. 13. [4 points] Eukaryotic DNA replication initiates replication at multiple origins of replication simultaneously. How is eukaryotic replication initiated to ensure simultaneous initiation at multiple origins? Pre-replication complex is formed at all origins (2 points), Coordinate activation of these complexes allows for simultaneous initiation at all origins (2 points). Also get credit for mentioning CDK enzymes off during formation of Pre-RC followed by CDK enzymes on to allow for coordinate activation, 14. [4 points] Describe the mechanistic difference that distinguishes the splicing of group I introns from that of nuclear mRNA introns. Ans: In the group I introns, the initial transesterification in the RNA chain occurs as the hydroxyl group of a solvent guanosine nucleotide makes a nucleophilic attack on the phosphodiester bond with no lariat being formed. In mRNA introns, the attacking species is the 2 ‘-hydroxyl group of an adenylate residue in the intron itself making a lariat.

In addition to DNA polymerase’s intrinsic 3′-5′ exonuclease activity, mismatch repair, and base excision repair, two other mechanisms for DNA repair include _(oligo)nucleotide excision repair_ and ____direct repair___. 16. [6 points] In the initiation of transcription in E. coli, the catalytic subunit of RNA polymerase first combines with the ___σ ______ subunit to form the RNA polymerase holoenzyme complex which then binds to the DNA promoter to form the ____closed_________ complex. Melting of the DNA duplex by the polymerase then forms the _____open________ complex. After elongation, termination of transcription can occur with a ρ-independent mechanism with the formation of a __RNA hairpin______ and ___AU RNA-DNA hybrid duplex_________. Alternatively in the ρ- dependent mechanism, a rut site is recognized by a ___ρ helicase______ which separates the RNA from the DNA template. 17. [2 points] Which of the following statements about the poly(A) tails that are found on most eukaryotic mRNAs are correct? A) They are added as preformed polyriboadenylate segments to the 3′ ends of mRNA precursors by an RNA ligase activity. B) They are encoded by stretches of polydeoxythymidylate in the template strand of the gene. C) They are added by RNA polymerase II in a template-independent reaction using ATP as the sole nucleotide substrate. D) They are added by polyadenylate polymerase using ATP as the sole nucleotide substrate. E) They are cleaved from eukaryotic mRNAs by a sequence specific endonuclease that recognizes the RNA sequence AAUAAA. Circle the correct answer. 18. [2 points] Which process listed below is common to the post-transcriptional modification of eukaryotic mRNA? A) Addition to the 5′ end of a 7-methylguanosine in a 5′,5′ triphosphate linkage. B) Addition to the 3′ end of a 7-methylguanosine in a 3′,5′ triphosphate linkage. C) Addition to the 5′ end of a 7-methyladenosine in a 5′,5′ triphosphate linkage. D) Methylation of ribose 3′-hydroxyl groups. E) Phosphorylation of ribose 2′-hydroxyl groups. Circle the correct answer. 19. [5 points] According to the wobble hypothesis, what is the minimum number of tRNAs required to decode the six arginine codons CGU, CGC, CGA, CGG, AGA, AGG?

Minimum number of tRNA molecules: ___3 (2 points)________ Indicate the tRNA anticodon sequences (correctly labeling 5′ and 3′ ends) that would recognize each of the six codons for this minimal number of tRNA molecules: Anticodon sequence (label polarity) Codon recognized 3′-GCI-5′ or 3′-GCG-5′ 5′-CGU-3′ 3′-GCI-5′ or 3′-GCG-5′ 5′-CGC-3′ 3′-GCI-5′ or 3′-GCU-5′ 5′-CGA-3′ 3′-GCC-5′ or 3′-GCU-5′ 5′-CGG-3′ 3′-UCU-5′ (not 3′-UCI-5′) 5′-AGA-3′ 3′-UCU-5′ (not 3′-UCC-5′) 5′-AGG-3′ 3 points for all correct, -0.5 for each incorrect or missing answer. okay if anticodons are inverted as long as ends are labeled correctly 20. [4 points] For the following statements about the aminoacyl-tRNA synthetase reaction, indicate whether the statement is true or false (by circling T or F). If the statement is false, provide a brief correction on the adjacent line. T or F GTP hydrolysis is involved in the reaction _____ATP is involved_____________ T or F An acyl thioester bond is formed _____An acyl ester is formed_____________ T or F The reaction occurs through two steps ______________________________ T or F The amino acid is attached to the 2′ hydroxyl of an adenosine at the 3′ end of the tRNA molecule ____3′ OH of this adenosine is attached to amino acid__________ 21. [5 points] E. coli cells are placed in a growth medium containing lactose. Please indicate how the following perturbations would effect the expression of the lactose operon (write either increase, decrease, or no change in the spaces provided). __Increase___ Reduction in the level of glucose in the media ___Increase__ Introduction of a mutation of the -35 and -10 region of the lac promoter to more closely resemble the σ70 consensus sequence ___Decrease_ Introduction of a mutation that inactivates lactose permease (product of lacY gene) ___Decrease_ Introduction of a mutation of adenylate cyclase that reduces cellular levels of cAMP ___Decrease__Introduction of a mutation in CRP protein to cause it to bind to the operator sequence

For the following statements about prokaryotic translation, indicate whether the statement is true or false (by circling T or F). If the statement is false, provide a brief correction on the adjacent line. T or F Amino acids are added to the amino terminus of the growing polypeptide chain _____added to c terminus_____________________ T or F Specific sequences within the mRNA ensure that translation begins at the correct codon ____________________________ T or F Peptide bonds form between an aminoacyl-tRNA and a peptidyl-tRNA positioned at the A and P sites, respectively, of the ribosome ______________________________ T or F Termination involves the binding of a terminator tRNA to a stop codon on the mRNA _____release factor binds to ribosome for termination___________ T or F Puromycin binds to the A site leading to hydrolysis of peptide from peptidyl-tRNA in the P site _an amide bond is formed between puromycin and peptide_ 23. [4 points] Give two examples of coactivators and explain how they are involved in transcriptional activation in eukaryotes. Your answer should include the identities of the binding partners of the coactivators. Two examples are TFIID and Mediator (2 points). They are involved in activation when bound to a transactivator where they help to recruit the basal transcription machinery and RNAP II to the promoter. (2 points)

For the following statements about the trp operon leader RNA region shown below, indicate whether each statement is true or false (by circling T or F). trp 1 2 3 4 5ʹ structural genes T or F A short open reading frame, containing Trp codons among others, exists within sequence 1 of the leader RNA T or F The structure of the leader RNA in vivo depends on the position of the ribosomes translating it. T or F Base pairing of sequence 1 and 2 blocks association of sequence 2 and 3, leading to attenuation T or F Base pairing between sequence 2 and 3 forms a stem loop that acts as a ρ-independent terminator T or F Covering of sequence 2 by ribosome when tryptophan levels are high prevents ρ-independent termination 25. [8 points] Shown to the right are ClaI: NotI: EcoRV the recognition sequences for several type II restriction endonucleases: A) Draw the structure of both ends of a linear DNA fragment that are produced by a ClaI restriction digest. (Label all 5′ and 3′ ends). AT-3′ and 5′-CGAT TAGC-5′ 3′-TA B) Draw the structure resulting from the reaction of the end sequences in A) with DNA polymerase and the four deoxynucleoside triphosphates. ATCG-3′ and 5′-CGAT TAGC-5′ 3′-GCTA C) Draw the structure of both ends of a linear DNA fragment that are produced by a EcoRV restriction digest. (Label all 5′ and 3′ ends). GAT-3′ and 5′-ATC CTA-5′ 3′-TAG D) Draw the structure of the two possible junctions produced if the ends with the structures in B) are ligated to the ends with the structures in C). (Label all 5′ and 3′ ends). 5′- GATCGAT -3′ and 5′- ATCGATC -3′ 3′- CTAGCTA -5′ 3′- TAGCTAG -5′

Which of the following partial amino acid sequences from a protein whose gene you wish to clone would be most useful in designing an oligonucleotide probe to screen a cDNA library? A) Met-LeuArg-Leu B) Met-Trp-Cys-Trp Explain why you selected your answer: B) is minimally degenerate so less oligonucleotides would need to be synthesized to find the gene. (1 point) 27. [4 points] A scientist wishes to produce a mammalian protein in E . coli. The protein is a glycoprotein with a molecular weight of 40,000. Approximately 20% of its mass is polysaccharide. The isolated protein is usually phosphorylated and contains three disulfide bonds. The cloned gene contains no introns. (a) What sequences or sites will be required in the vector to get this gene regulated, transcribed, and translated in E. coli? The cloned gene must be preceded by a good E. coli promoter and its associated operator and by a ribosome-binding (Shine-Dalgarno) sequence. The other end of the gene should have a transcription terminator sequence. (b) List two problems in E. coli that might arise in producing a protein identical to that isolated from mammalian cells and describe each problem in no more than two sentences. Potential problems are that (1) E. coli enzymes may not glycosylate the protein, which may affect its folding and activity, and (2) the protein kinases that phosphorylate the protein in mammalian cells are probably absent in E. coli ; therefore, the engineered protein will not be phosphorylated.

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