Babel fish. Why is protein synthesis also called translation?

Careful, but not too careful. Why is it crucial that protein synthesis has an error frequency of 10⁻⁴?

Commonalities. What features are common to all tRNA molecules?

The ol’ two step. What two reaction steps are required for the formation of an aminoacyl-tRNA?

The same but different. Why must tRNA molecules have both unique structural features and common structural features?

Charge it. In the context of protein synthesis, what is meant by an activated amino acid?

Synthetase mechanism. The formation of isoleucyl-tRNA proceeds through the reversible formation of an enzyme-bound Ile-AMP intermediate. Predict whether ³²P-labeled ATP is formed from ³²PP_i when each of the following sets of components is incubated with the specific activating enzyme:

1 = 2, for sufficiently large values of 1. The energetic equivalent of two molecules of ATP is used to activate an amino acid, yet only one molecule of ATP is used. Explain.

Sieves. Using threonyl-tRNA synthetase as an example, account for the specificity of threonyl-tRNA formation.

Use all available information. Suggest a reason why there are two classes of aminoacyl-tRNA synthetases, with each class recognizing a different face of the tRNA.

Going wobbly. Explain how it is possible that some tRNA molecules recognize more than one codon.

Light and heavy ribosomes. Ribosomes were isolated from bacteria grown in a “heavy” medium (¹³C and ¹⁵N) and from bacteria grown in a “light” medium (¹²C and ¹⁴N). These 60S 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 70S ribosomes would you expect to see in the density gradient?

The price of protein synthesis. 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 hydrolysis of PP_i is equivalent to the hydrolysis of ATP for this calculation.

Correct phasing. What is meant by the phrase reading frame?

Suppressing frameshifts. The insertion of a base in a coding sequence leads to a shift in the reading frame, which in most cases produces a nonfunctional protein. Propose a mutation in a tRNA that might suppress frameshifting.

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Tagging a ribosomal site. Design an affinity-labeling reagent for one of the tRNA-binding sites in E. coli ribosomes.

Viral mutation. An mRNA transcript of a T7 phage gene contains the base sequence

Predict the effect of a mutation that changes the red G to A.

A new translation. A transfer RNA with a UGU anticodon is enzymatically conjugated to ¹⁴C-labeled cysteine. The cysteine unit is then chemically modified to alanine. The altered aminoacyl-tRNA is added to a protein-synthesizing system containing normal components except for this tRNA. The mRNA added to this mixture contains the following sequence:

5′–UUUUGCCAUGUUUGUGCU–3′

What is the sequence of the corresponding radiolabeled peptide?

Two synthetic modes. Compare and contrast protein synthesis by ribosomes with protein synthesis by the solid-phase method (Section 3.4).

Triggered GTP hydrolysis. 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?

Blocking translation. 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.

Directional problem. 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 A₁, A₂, A₃, A₄, and A₅. Peptide A₁ is the amino-terminal peptide, and A₅ 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 ¹⁴C label.

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

A timing device. 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?

Not just RNA. What are the roles of the protein factors required for protein synthesis?

Membrane transport. What four components are required for the translocation of proteins across the endoplasmic reticulum membrane?

Push. Don’t pull. What is the energy source that powers the cotranslational movement of proteins across the endoplasmic reticulum?

You have to know where to look. Bacterial messenger RNAs usually contain many AUG codons. How does the ribosome identify the AUG specifying initiation?

Fundamentally the same, yet … List the differences between bacterial and eukaryotic protein synthesis.

Like a border collie. What is the role of the signal-recognition particle in protein translocation?

An assembly line. Why is the fact that protein synthesis takes place on polysomes advantageous?

Match ‘em

Wasted effort? Transfer RNA molecules are quite large, given that the anticodon consists of only three nucleotides. What is the purpose of the rest of the tRNA molecule?

Evolutionary amino acid choice. 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.)

Contrasting modes of elongation. The two basic mechanisms for the elongation of biomolecules are represented in the adjoining illustration. In type 1, the activating group (X) is released from the growing chain. In type 2, the activating group is released from the incoming unit as it is added to the growing chain. Indicate whether each of the following biosyntheses is by means of a type 1 or a type 2 mechanism:

Enhancing fidelity. Compare the accuracy of DNA replication, RNA synthesis, and protein synthesis. Which mechanisms are used to ensure the fidelity of each of these processes?

Déjà vu. Which protein in G-protein cascades plays a role similar to that of elongation factor Ts?

Family resemblance. Eukaryotic elongation factor 2 is inhibited by ADP ribosylation catalyzed by diphtheria toxin. What other G proteins are sensitive to this mode of inhibition?

The exceptional E. coli. In contrast with E. coli, most bacteria do not have a full complement of aminoacyl-tRNA synthetases. For instance, Helicobacter pylori, the cause of stomach ulcers, has tRNA^Gln, but no Gln-tRNA synthetase. However, glutamine is a common amino acid in H. pylori proteins. Suggest a means by which glutamine can be incorporated into proteins in H. pylori. (Hint: Glu-tRNA synthetase can misacylate tRNA^Gln.)

The final step. What aspect of primary structure allows the transfer of linear nucleic acid information into the functional three-dimensional structure of proteins?

Helicase helper. The initiation factor eIF-4 displays ATP-dependent RNA helicase activity. Another initiation factor, eIF-4H, has been proposed to assist the action of eIF-4. Graph A shows some of the experimental results from an assay that can measure the activity of eIF-4 helicase in the presence of eIF-4H.

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Size separation. The protein-synthesizing machinery was isolated from eukaryotic cells and briefly treated with a low concentration of RNase. The sample was then subjected to sucrose gradient centrifugation. The gradient was fractionated and the absorbance, or optical density (OD), at 254 nm was recorded for each fraction. The following plot was obtained.