1,600 (4 × 400) phosphoanhydride bonds. Typically, four ATP/GTP molecules are hydrolyzed per amino acid incorporated into protein.

(a) Amide or peptide. (b) Ester. (c) Phosphodiester. (d) Hydrogen bonds. (e) Noncovalent bonds, including hydrogen bonds and van der Waals forces.

The 15,000 ribosomes in an E. coli cell can synthesize more than 29,000 proteins in 20 minutes.

Using polysomes, the cell can produce several protein molecules on a single mRNA molecule. Because mRNAs have an average lifetime of just a few minutes in the cell, polysomes maximize the number of proteins that can be made per unit time.

The fMet-tRNA^fMet binds directly to the P site during initiation of protein synthesis, positioned there to make the first peptide bond with a second aminoacylated tRNA that is positioned in the A site.

S-18

Proline is incorporated during protein synthesis; posttranslational processing adds the hydroxyl group.

Isoleucyl-tRNA synthetase sometimes catalyzes the addition of valine to tRNA^Ile; valine is similar to but smaller than isoleucine and can readily fit into the synthetase active site. Histidine has no close structural analogs among the amino acids, greatly lowering the chance that tRNA^His will be charged with the incorrect amino acid.

Yes. In polypeptide synthesis, removal of the last amino acid added (by hydrolytic cleavage of the last peptide bond to form) would sever the covalent link between the polypeptide and the tRNA in the ribosomal P site. This would terminate polypeptide synthesis.

IF-2: the 70S ribosome would form, but initiation factors would not be released and elongation could not start. EF-Tu: the second aminoacyl-tRNA would bind to the ribosomal A site, but no peptide bond would form. EF-G: the first peptide bond would form, but the ribosome would not move along the mRNA to vacate the A site for binding of a new EF-Tu–tRNA.

(a) The synthetase recognizes the G–U base pair (between G3 and U70) in the amino acid arm of tRNA^Ala. (b) The mutant tRNA would insert Pro residues at codons that specify Ala. (c) A mutation in tRNA^Pro that allowed it to be recognized and aminoacylated by Ala-tRNA synthetase would have similar effects. (d) Such changes would insert Pro residues at many inappropriate sites in polypeptides, inactivating many proteins, and thus would be lethal.

A tmRNA (transfer-messenger RNA) binds to the A site of the stalled ribosome. Part of its structure acts as an aminoacylated tRNA^Ala, leading to transfer of an Ala residue to the C-terminus of the partially synthesized protein. Another part of the tmRNA structure functions as an mRNA, permitting the addition of 10 additional amino acids to the protein before a stop codon is reached. After the polypeptide is released from the ribosome, the 10 amino acid residues incorporated at its C-terminal end act as a signal for degradation by cellular proteases.

An unknown location, but not the nucleus. The protein will be bound by SRP and transported as it is synthesized into the ER lumen. Its fate from there would depend on other signals. The NLS would not be used, because it would not be accessible to the cytosolic proteins that normally bind it.

Chloramphenicol inhibits bacterial protein synthesis and mitochondrial protein synthesis. The effects on mitochondrial ribosomes give rise to the human toxicity.

(a) Poly(UG) generates polypeptides containing Phe, Leu, Val, Cys, Trp, and Gly. (b) Ala is not normally present in polypeptides synthesized in response to poly(UG). (c) Yes. In both cases, about one-third of the added label is incorporated into the product polypeptide, although the yield is slightly reduced in the Raney nickel–treated preparation. (d) This experiment was a control to confirm that Raney nickel treatment did not have a general deleterious effect on tRNA. (e) In most scientific studies, it is useful to confirm a result by two or more different methods. The extra experiment rendered the final conclusion unambiguous.

(a) The 50S subunit contains the peptidyl transferase activity of the ribosome. It includes the parts of the P and A sites that interact with the 3′ ends of charged tRNAs. (b) The CCA sequence at the 3′ end of the hexanucleotide is present at the 3′ end of every tRNA and is required for specific binding to the 50S subunit. (c) The fMet- oligonucleotide must bind in the P site, and the puromycin in the A site. (d) The reaction is much simpler and does not rely on added protein factors such as initiation and elongation factors, which would be inactivated by the protein removal treatments. (e) Given the capacity of T. aquaticus to grow at high temperatures, the rRNA in the 50S subunits might be particularly stable and able to withstand protein removal. (f) The result in Figure 2 shows that chloramphenicol and carbomycin strongly inhibit the reaction, so the reaction is indeed catalyzed by the peptidyl transferase activity of the 50S subunit. In addition, the RNA component of the subunit is essential for activity. (g) The protein extraction procedures were thorough, but there was a possibility that small amounts of protein remained associated with the 23S rRNA.