Biochemistry 302. Exam 2. March 10, Answer Key

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1 1 Biochemistry 302 Exam 2 March 10, 2004 Answer Key

2 2 Biochemistry 302, Spring 2004 Exam 2 (100 points) Name I. Short answer 1. Identify the 5 end, 3 end, amino acid acceptor nucleoside, and bases comprising the anticodon loop on this cloverleaf model of trna (4 points). 2. Write the general chemical equations that describe the sequential two-step reaction catalyzed by aminoacyl trna synthetases (2 points). Amino acid + ATP Aminoacyl-adenylate (AMP) + PPi Aminoacyl-adenylate + trna Aminoacyl-tRNA + AMP 3. In which ribosomal subunit(s) does the acceptor stem and the anticodon loop of a charged trna reside (2 points)? Acceptor stem large subunit Anticodon loop small subunit 4. Where exactly does the amino acid get attached on the trna acceptor nucleoside (i.e. base or ribose)? Be specific and name the substituent group (2 points). 3 hydroxyl of adenosine acceptor 5. What types of intramolecular interactions stabilize the tertiary structure of trna (2 points)? triple-base (H-bonding), base-stacking (van der Waals), and base-phosphate backbone interactions

3 3 6. This modified amino acid plays a special role in information metabolism in prokaryotes. Name the amino acid and describe its unique functional role (4 points). N-formylmethionine (fmet), initiation of protein synthesis (fmet-trna is the first charged trna to associate with mrna template and 30S subunit) 7. i) Explain the chemical role of the N3 group of adenine in this reaction scheme. ii) Name the macromolecular complex capable of catalyzing the reaction as described. iii) Describe what the products of this reaction are. (6 points) i) general base: as an imino tautomer, N3 attracts a proton from α-amino group facilitating nucleophilic attack of this amino group on the carbonyl carbon ii) large ribosomal subunit iii) de-acylated trna (trna-oh) and a peptidyl-trna with one additional amino acid 8. Name the enzyme that catalyzes the polynucleotide synthesis reaction as indicated (2 points). Reverse transcriptase

4 4 II. Multiple choice. Write the letter of the best answer on the line provided (3 points each). 1. _A_ The sequence, AAUAAA, in the 3 -UTR of a eukaryotic mrna sequence is a signal for: A. Cleavage and polyadenylation of the 3 end B. Translational initiation C. Transcriptional attenuation D. None of the above 2. _D_ Which of the following enzymes are involved in post-transcriptional modifications (i.e. capping reactions) on the 5 end of eukaryotic mrnas? A. Guanylyl transferase B. Guanine-7-methyl transferase C. 2 -O-methyl transferase D. All of the above 3. _B_ Processing of ribosomal RNA and transfer RNA in prokaryotes is carried out by: A. Proteases B. Ribonucleases C. Group II introns D. Spliceosome 4. _A_ This modified base is found at the 5 -position in the anticodon of trna Arg, forms wobble base pairs, and is generated by the action of adenosine deaminase: A. Inosine B. Methylcytosine C. Ribothymidine D. Pseudouracil 5. _A_ The DNA recognition helices of helix-turn-helix proteins such as λ repressor and zincfinger proteins such as Zif268 are related because: A. They form chemical contacts in the major groove of B-DNA B. They form chemical contacts in the minor groove of A-DNA C. They preferentially interact with Z-DNA forming sequences 6. _C_ In the absence of a mrna template, dissociation of ribosomal subunits is facilitated by: A. Specific translation initiation factors B. Intracellular ionic strength conditions C. Both A and B D. Neither A or B

5 5 7. _A_ The Sanger method of DNA sequencing relies on chain termination chemistry mediated by which of the following DNA synthesis inhibitors: A. 2, 3 -dideoxy NTPs B. EDTA C. 3 -O-methylated NTPs 8. _C_ The antibiotics, puromycin and streptomycin, are inhibitors of: A. DNA synthesis B. RNA synthesis C. Protein synthesis D. Both A and B 9._B_ Binding of an inducer such as lactose or IPTG to the lac repressor protein promotes a conformational change in the protein that: A. Enhances DNA-binding affinity of lac repressor for operator sequences B. Reduces DNA-binding affinity of lac repressor for operator sequences C. Stimulates proteolytic degradation of lac repressor 10._D_ Attenuation (i.e. premature transcriptional termination) of the E. coli biosynthetic trp operon occurs by a mechanism that is dependent upon: A. Ability of the 5 -leader sequence to adopt a specific stem-loop structure that promotes factor-independent transcriptional termination B. Ribosome positioning along the 5 leader sequence C. Relative intracellular concentration of tryptophan D. All of the above 11._A_ Synergistic activation of gene expression in higher eukaryotes is generally mediated by: A. Multiple trans-acting factors with distinct cis-element binding specificities functioning in a cooperative manner to recruit RNA polymerase II, associated general transcription factors, and/or chromatin remodeling complexes to a gene promoter B. Single gene-specific trans-acting factor functioning in an autonomous manner to recruit RNA polymerase II, associated general transcription factors, and/or chromatin remodeling complexes to a gene promoter C. Targeted recruitment of the ribosome to a specific mrna template 12._D_ The composition of the eukaryotic ribosome differs from the prokaryotic ribosome because the eukaryotic ribosome has: A. An additional 5.8 S rrna component in the large subunit B. More proteins in the small subunit C. More proteins in the large subunit D. All of the above

6 6 III. True/False. Write T or F on the line provided. For statements that are deemed false, provide a brief justification for your answer. (2 points each) 1. Excision of introns from nuclear pre-mrnas can occur in the absence of protein and is completely RNA-dependent. _F_ need snrnps 2. Group I introns require a guanosine cofactor to initiate self-splicing. _T_ 3. Stem-loop helices in trna molecules exist primarily in a B-form configuration. _F_ RNA duplexes are usually A-form. 4. The restriction enzyme, Dpn I, is often used in mutagenesis applications because of its ability to digest methylated DNA templates. _T_ 5. Guanine nucleotide exchange factors such as EF-Ts (prokaryotic) and eif2b (eukaryotic) initiate exchange by virtue of their intrinsic GTPase activity. _F_ Protein-protein interaction-mediated conformational changes promote exchange. 6. Messenger RNA synthesis and processing reactions occur as independent molecular events in the nucleus of eukaryotes. _F_ Biochemical evidence suggests that factors involved in splicing and polyadenylation can associate with RNA polymerase. 7. Despite differences in primary nucleotide sequence, the stem-loop or secondary structure of 16S rrna is highly conserved across all kingdoms of life. _T_ 8. RNA strand cleavage by the hammerhead ribozyme involves formation of an intramolecular 2-3 phosphodiester linkage. _T_ IV. Answers requiring a carefully worded sentence or two (6 points each). 1. Describe the molecular basis of ATP selection by poly(a) polymerase. In other words, why does the enzyme recognize ATP but not other NTPs? The organization of the active site of PAP is such that only ATP can fit properly. The monocyclic pyrimidine ring present in CTP and UTP is two small to H-bond with certain amino acid side chains (Asp) while the bicyclic purine ring of GTP possesses substituent groups (attached to C6 and C2) which preclude formation of stabilizing van der Waals, H-bond, and metal ion-facilitated contacts that can take place with active site-bound ATP.

7 7 2. Explain the chemical role of the branch point adenosine in nuclear pre-mrna splicing. The branch point adenosine (BPA) initiates the first of two trans-esterification reactions required for intron removal. The 2 hydroxyl (OH) of the BPA attacks a phosphate at the 5 intron/exon boundary (i.e. 5 splice site) resulting in formation of 1) a partially excised loop-containing intron stabilized by a 2-5 phosphodiester bond and 2) a 5 exon with a free 3 OH to initiate the second transesterification reaction on the 3 splice site. 3. Describe the location, sequence characteristics, and function of Shine-Dalgarno elements in prokaryotic mrnas. Shine-Dalgarno elements are short purine-rich sequences that are located upstream (5 ) and relative close to the AUG start codon in a prokaryotic mrna template. These elements assist in alignment of the small ribosomal subunit by virtue of base pairing with the 3 end of 16S rrna. (They also serve as recognition elements for the fmettrna/if2 initiator complex.) 4. How can chromatin (nucleosome) remodeling contribute to either activation or repression of gene expression? Swi/Snf ATPase complexes and histone acetyltransferases promote disassembly, sliding, and/or removal of histones from the DNA, which can expose cis-regulatory elements to recognition by transcriptional activators. On the other hand, histone deacetylases promote deposition of histones onto DNA, which could act as a barrier to target site recognition by transcriptional activators resulting in repression. V. Propose a plausible model for how the 21 st amino acid, selenocysteine (SeC), is selectively incorporated into a peptide chain using the UGA stop codon. Hint: All mrna templates encoding SeC-containing redox enzymes contain a specific stem-loop signal in the 3 UTR. (up to 5 bonus points) Mechanism must necessarily involve recoding of the UGA stop codon to a SeC insertion codon. So some other cis-acting element in the mrna template and probably an associated trans-acting factor(s) must somehow signal the translation machinery to recognize the UGA as a SeC insertion site rather than a stop site. It so happens that mrnas encoding selenoproteins all contain a highly conserved stem-loop structure in their 3 UTRs. This cis-element known as SECIS (for selenocysteine insertion sequence) serves as a binding site for a ribonucleoprotein complex containing SeC-tRNA SeC and a special elongation factor that helps deliver the SeC-tRNA to the ribosome as it encounters the UGA codon.