Mol Bio Ps-10

Molecular Biology, Spring 2006 Problem Set #10 (Lectures 17 & 18) 1) The genetic code was deciphered in part by experiments in which polynucleotides of repeating sequences were used as mRNAs to direct protein synthesis in cell-free extracts. In this system, ribosomes can begin protein synthesis anywhere along the RNA molecule, without the need for a start codon. What types of polypeptides would you expect to be synthesized if the following polynucleotides were used as templates in such a cell-free extract? a) UUUUUUUUU b) AUAUAUAUA c) AUCAUCAUC I couldn’t figure this one out 2) One remarkable feature of the genetic code is that amino acids with similar chemical properties (e.g. hydrophobicity) often have similar codons. Can you suggest a possible explanation for this phenomenon as it relates to the protein synthesis machinery? In case of mutation then the most similar aa gets translated next 3) A mutation in a bacterial gene generates a UGA stop codon in the middle of the mRNA coding region. A second mutation in the cell leads to a single nucleotide change in a tRNA that allows the correct translation of the protein; that is, the second mutation “suppresses” the defect caused by the first. The altered tRNA translates the UGA as tryptophan. What nucleotide change has probably occurred in the mutant tRNA molecule. What consequences would the presence of such a mutant tRNA have for the translation of normal genes in the cell? He did this one in class and this is only what I got from it---Wobble position can read through stop codons. Wobble position can allow for reading of multiple codons 4) In a clever experiment performed in 1962, a cysteine that was already attached to tRNACys was chemically converted to alanine. These alanyl-tRNACys molecules were then added to a cell-free translation system from which normal tRNACys molecules had been removed. When the resulting protein was analyzed, it was found that alanine had been inserted at every point in the protein chain where cysteine was supposed to be. What does this experiment tell you about the role of aminoacyl-tRNA synthetases during the normal translation of the genetic code. 5) One strand of a section of DNA isolated from E. coli reads: 5’ GTAGCCTACCCATAGG 3’ a) Suppose that an mRNA is transcribed from this DNA using the complementary strand as a template. What is the mRNA sequence in this case? CATCGGATGGGTATCC template from DNA GUAGCCUACCCAAGG mRNA

b) How many different polypeptides could be potentially be made from this sequence of RNA? 3 Would the same peptides be made if the other strand of DNA served as the template for transcription? no c) What peptide would be made if translation started exactly at the 5’ end of the mRNA in part A? ala When tRNAAla leaves the ribosome, what tRNA will be bound next? In cass he said proline?? When the amino group of alanine forms a peptide bond, what bonds, if any, are broken and what happens to the tRNAAla? Break amino-acyl bonds and tRNA exits out E site d) Suppose this stretch of DNA is transcribed as indicated in part A, but you do not know which reading frame is to be used. Could this DNA segment originate from the beginning of the coding region of a gene? No bc no met The middle? yes The end?? yes How can you tell? 6) Polycistronic mRNAs are common in prokaryotes, but extremely rare in eukaryotes. Describe the key differences in protein synthesis that underlie this observation. Prok—polycistronicsingle mRNA can encode multiple proteins. Ex) trp operon--translation occurs at 5 diff start sites on the same mRNA which yields 5 diff proteins Euk—trp operon---the 5 proteins are located on 5 diff chromosomes. Each chromosome is translated at its own promoter site to yield the 5 diff proteins 7) You have isolated an antibiotic, named edeine, from a bacterial culture. Edeine inhibits protein synthesis but has no effect on either DNA synthesis or RNA synthesis. When added to cell lysates, edeine stops protein synthesis after a short lag. By contrast, cycloheximide stops protein synthesis immediately. Analysis of the edeine-inhibited lysate showed that no polyribosomes remained by the time synthesis had stopped. Instead, all the mRNA was found bound to the small ribosomal subunit, which contained equimolar amounts of initiator tRNA. a) What step in protein synthesis does edeine inhibit? Prevents lg subunit binding small subunit b) Why is there a lag between the addition of edeine and cessation of protein synthesis???? What determines the length of the lag? Length of lag is relative to the amt of message c) Why are polyribosomes absent after edeine treatment? The large and small subunits are blocked he did this in class too so this was all I got from it 8) In cell lysates, the polynucleotide 5’ AUGUUUUUUUUU 3’ directs the synthesis of Met-Phe-Phe-Phe. In the presence of farsomycin, a new antibiotic perfected by Fluhardy

Pharmaceuticals, this polymer directs the synthesis of Met-Phe only. From this information, which of the following deductions could you make about faromycin? a) It prevents formation of the 80S initiation complex, which contains the initiator tRNA and both ribosomal subunits. b) It inhibits binding of aminoacyl-tRNAs to the A-site of the ribosome c) It inactivates peptidyl transferase activity of the large ribosomal subunit d) It blocks translocation of peptidyl-tRNA from the A-site to the P-site of the ribosome e) It interferes with chain termination and the release of the peptide