Chapter 17, Problem 19P

J.T. Lis and collaborators have developed an experimental protocol called PRO-Seq that pinpoints all the positions in a genome at which transcriptionally engaged RNA polymerase (that is, enzyme molecules that are actively in the process of synthesizing RNA) is located at a specific time. The accompanying figure shows the results of a PRO-Seq analysis of one region of the human genome, in the vicinity of the Dnaj4 gene. Vertical red lines show the location of transcriptionally engaged RNA polymerase that is moving along the DNA in the Section 17.3 left-to-right direction, while vertical blue lines show transcriptionally engaged RNA polymerase in the opposite direction. The length of a line indicates the frequency at which active polymerase is found at that position along the genome in the sample.

The two samples were taken from the same culture of human cells grown on a petri plate. The culture was grown under normal conditions and then sampled (Before Heat Shock); then the culture was grown at high temperature and sampled one hour later (After Heat Shock).

a.	Transcriptionally engaged RNA polymerase is not uniformly distributed along the gene. What does this fact signify?
b.	The data, along with your answer to part (a), together suggest that the binding of RNA polymerase to the promoter is not the only rate-limiting step in the transcription of the Dnaj4 gene, whether before or after heat shock. What other step is involved? Which step appears to be most directly regulated by heat shock?
c.	When RNA polymerase binds to the promoter of this gene, does it know which strand of DNA to use as the template?
d.	Where does the transcription of the DNAj4 gene end? Do the data clearly show the existence of a single, well-defined transcription stop site?
e.	How do you think that the PRO-Seq method can localize transcriptionally engaged RNA polymerase specifically as opposed to RNA polymerase that might bind to DNA but is not catalyzing transcription?