1.  Babel fish. Why is protein synthesis also called translation?

2.  Correct phrasing. What is meant by the phrase reading frame?

3.  Match ’em. Match each term on the right with the terms in parts a, b, and c. ✓ 3 ✓ 4

4.  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? ✓ 3

5.  Light and heavy ribosomes. Density-gradient centrifugation is a technique that allows the separation of biological molecules and molecular complexes by differences in density. 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 70S ribosomes were added to an in vitro system undergoing protein synthesis. An aliquot removed several hours later was analyzed by density-gradient centrifugation. How many types of 70S ribosomes differing in density would you expect to see in the density gradient? ✓ 4 ✓ 5

6.  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.

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



Predict the effect of a mutation that changes the G identified by the arrow to A. ✓ 3

8.  Enhancing fidelity. Compare the accuracy of (a) DNA replication, (b) RNA synthesis, and (c) protein synthesis. What mechanisms are used to ensure the fidelity of each of these processes? ✓ 4

9.  You have to know where to look. Bacterial messenger RNAs usually contain many AUG codons. How does the ribosome identify the AUG specifying initiation? ✓ 4 ✓ 5

10.  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? ✓ 4

11.  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. ✓ 3

12.  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. ✓ 4

(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 heaviest to lightest?

(c) What does this experiment tell you about the direction of protein synthesis?

13.  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? ✓ 4

14.  Fundamentally the same, yet … List the differences between bacterial and eukaryotic protein synthesis. ✓ 6

15.  Membrane transport. What four components are required for the translocation of proteins across the endoplasmic reticulum membrane? ✓ 4

16.  Like a border collie. What is the role of the signal-recognition particle in protein translocation? ✓ 4 ✓ 5

17.  Push, don’t pull. What is the energy source that powers the cotranslational movement of proteins across the endoplasmic reticulum? ✓ 5

18.  An assembly line. Why is the fact that protein synthesis takes place on polysomes advantageous? ✓ 5

19.  Iron regulation. What effect would you expect from the addition of an iron-response element (IRE) to the 5′ end of a gene that is not normally regulated by iron levels? To the 3′ end? ✓ 7

20.  Predicting microRNA regulation. Suppose that you have identified an miRNA that has the sequence 5′-GCCUAGCCUUAGCAUUGAUUGG-3′. Propose a strategy for identifying mRNA that might be regulated by this miRNA, given the sequences of all mRNAs encoded by the human genome. ✓ 7

21.  Déjà vu. Which protein in G-protein cascades plays a role similar to that of elongation factor Ts? ✓ 4

22.  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?

23.  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:

(a) Glycogen synthesis

(b) Fatty acid synthesis

(c) C₅ → C₁₀ → C₁₅ in cholesterol synthesis

(d) DNA synthesis

(e) RNA synthesis

(f) Protein synthesis

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

25.  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. ✓ 4



(a) What are the effects on eIF-4 helicase activity in the presence of eIF-4H?

(b) Why did measuring the helicase activity of eIF-4H alone serve as an important control?

(c) The initial rate of helicase activity of 0.2 μM of eIF-4 was then measured with varying amounts of eIF-4H (graph B). What ratio of eIF-4H to eIF-4 yielded optimal activity?



(d) Next, the effect of RNA–RNA helix stability on the initial rate of unwinding in the presence and absence of eIF-4H was tested (graph C). How does the effect of eIF-4H vary with helix stability?



(e) How might eIF-4H affect the helicase activity of eIF-4A?

[Data from N. J. Richter, G. W. Rodgers, Jr., J. O. Hensold, and W. C. Merrick. Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H. J. Biol. Chem. 274:35415–35424, 1999.]

26.  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 plot in graph A was obtained. ✓ 4



(a) What do the three peaks of absorbance in graph A represent?

The experiment was repeated except that, this time, the RNase treatment was omitted.

(b) Why is the centrifugation pattern in graph B more complex? What do the series of peaks near the bottom of the centrifuge tube represent?



Before the isolation of the protein-synthesizing machinery, the cells were grown in low concentrations of oxygen (hypoxic conditions). Again the experiment was repeated without RNase treatment (graph C).



(c) What is the effect of growing cells under hypoxic conditions?

[Data from M. Koritzinsky et al. EMBO J. 25:1114–1125, 2006.]

27.  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.

28.  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.) ✓ 4