Transcription Regulation by Initiating NTP Concentration: rRNA Synthesis in Bacteria Gaal et al., Science, 278: 2092 (Dec 19, 1997).
1. What is the phenomenon of growth rate control? Why is it important?
2. What are the different known regulatory elements present in the rrnB promoter? Was it reasonable for the authors to use the -41 template?
3. Why was it important that for the three promoter templates tested, each had the same transcribed sequence?
4. Why might salt concentrations and the state of the template (i.e. supercoiled versus linear) affect the absolute concentration of ATP required for maximal transcription?
5. What conclusion can you draw about transcriptional initiation based on the data in Fig. 2?
6. What is the experimental evidence that the authors present that indicates that the initiating nucleotide is specifically important for in vitro transcription from the rrn P1 promoters?
7. What is the evidence that purine nucleotide concentrations in vivo affect growth rate control?
8. Exactly how is open complex formation measured? What is the purpose of heparin? What are the two competing reactions that remain after heparin addition? What is the assumption about the relative rates of these two competing reactions?
9. How is the half life of the complex derived from the graph in Fig. 4? Why is the Y axis plotted the way it is?
10. How might the authors have carried out the genetic screen for mutants that affect growth rate dependence of rRNA transcription. Tucked away at the back of footnote 35 is the statement that "the mutant RNA polymerase also made less stable complexes with non-rRNA promoters." Does this influence your thinking about what this mutant perturbs and what its properties mean?
11. In the model proposed by the authors, what step(s) does [ATP] or [GTP] affect?
12. Based on the model the authors propose, is it important (or merely fortuitous) that the rrn P1 promoters all initiate with ATP or GTP? How does this lead to homeostasis of growth rate and protein synthesis?
13. What other observations does the model explain? How do the authors get around the observations that are not consistent with their model?
14. How might the sequence of a promoter affect stability of the open complex? How might you test your hypothesis?
15. What other experiments might you carry out to test the authors’ model?