Chapter 30

Ribosomes were isolated from bacteria grown in a "heavy" medium \(\left(^{13} \mathrm{C} \text { and }^{15} \mathrm{N}\right)\) and from bacteria grown in a "light" medium \(\left(^{12} \mathrm{C} \text { and }^{14} \mathrm{N}\right)\). These \(60 \mathrm{S}\) ribosomes were added to an in vitro system engaged in protein synthesis. An aliquot removed several hours later was analyzed by density-gradient centrifugation. How many bands of \(70 \mathrm{S}\) ribosomes would you expect to see in the density gradient?

What is the smallest number of molecules of ATP and GTP consumed in the synthesis of a 200 residue protein, starting from amino acids? Assume that the hy. drolysis of \(\mathrm{PP}_{\mathrm{i}}\) is equivalent to the hydrolysis of ATP for this calculation.

Compare the accuracy of (a) DNA replication, (b) RNA synthesis, and (c) protein synthesis. Which mechanisms are used to ensure the fidelity of each of these processes?

Ribosomes markedly accelerate the hydrolysis of GTP bound to the complex of EF-Tu and aminoacyl-tRNA. What is the biological significance of this enhancement of GTPase activity by ribosomes?

Devise an experimental strategy for switching off the expression of a specific mRNA without changing the gene encoding the protein or the gene's control elements.

Suppose that you have a protein-synthesis system that is synthesizing a protein designated A. Furthermore, you know that protein A has four trypsin- sensitive sites, equally spaced in the protein, that, on digestion with trypsin, yield the peptides \(A_{1}, A_{2}, A_{3}, A_{4},\) and \(A_{5} .\) Peptide \(A_{1}\) is the amino-terminal peptide, and \(A_{5}\) is the carboxyl- terminal peptide. Finally, you know that your system requires 4 minutes to synthesize a complete protein \(A .\) At \(t=0,\) you add all 20 amino acids, each carrying a \(^{14} \mathrm{C}\) label. (a) At \(t=1\) minute, you isolate intact protein A from the system, cleave it with trypsin, and isolate the five peptides. Which peptide is most heavily labeled? (b) At \(t=3\) minutes, what will be the order of the labeling of peptides from greatest to least? (c) What does this experiment tell you about the direction of protein synthesis?

Aminoacyl-tRNA synthetases are the only component of gene expression that decodes the genetic code. Explain.

EF-Tu, a member of the G-protein family, plays a crucial role in the elongation process of translation. Suppose that a slowly hydrolyzable analog of GTP were added to an elongating system. What would be the effect on the rate of protein synthesis?

Ornithine is structurally similar to lysine except ornithine's side chain is one methylene group shorter than that of lysine. Attempts to chemically synthesize and isolate ornithinyl-tRNA proved unsuccessful. Propose a mechanistic explanation. (Hint: Six-membered rings are more stable than seven- membered rings).