Discussion Paper: Moazed & Noller, Nature 342:142, 1989

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1. METHODS:
a. What was the basis for the assay used in this paper for binding of the various tRNA species to ribosomes?
What are the strengths and limitations of this method?
b. From what property of reverse transcriptase do the bands in the K lanes of the Figures result?
c. How could (and, presumably, were) the reverse transcriptase reactions internally standardized? For example, in Figure 3e, lanes 1-5, what other information is implicit or needed for comparison between the lanes?

2. For what is the N-acetyl-phe-tRNA an analog, and why?

3. What conclusions were drawn from the data shown in Figure 1?
In the absence of GTP or elongation factors, where does the energy for the P/P to P/E movement come from?

4. What experiment was done to determine whether the relative movement of N-acetyl tRNA from P to P/E state in the presence of puromycin was typical of normal ribosome function?

5. Figure 2, lanes 2 and 4. Also Fig 1e. What arguments would (could) you make against the hypothesis that the differences between these lanes were simply attributable to the presence or absence of steric blocking by the N-acetyl-phe on the N-acetyl-phe-tRNA relative to the deacylated tRNA, rather than site movement?

6. What was the method employed to construct "pre-translocation: ribosome complexes, and the rationale that the complex used does indeed represent the "pre-translocation" state?

7. By what criteria did the authors test whether the P site footprint in lane 4 of Fig 3 was attributable to deacylated tRNA?

8. What were the significant ways in which the new data provided in this paper differed from the expectations form the "classical" model of ribosome function?

9. What other methods might be used to demonstrate that the peptidyl end of the tRNA might occupy more than one site on the large subunit?

10. How would you distinguish between involvement of the tRNA ends relative to a fixed ribosome structure (Figure 5 c-e) versus relative movement of the ribosomal subunits (Figure 5 f-h)?

11. The results suggest a somewhat dynamic view of ribosome structure. What mechanisms might be used to alter ribosome conformation, particularly in the absence of energy? How might such mechanisms be tested?

12. What were the significant ways in which the new data provided in this paper differed from the expectations from the "classical" model of ribosome function?