IN VITRO ANALYSIS OF TRANSCRIPTION INITIATION AND PAUSING UPSTREAM OF THE ILVGMEDA OPERON

DEEPAK KUMAR AGRAWAL, Purdue University

Abstract

I have conducted in vitro experiments to explore the interaction between RNA polymerase and the promoters upstream of ilvG. The tandem promoters, p 1 and p 2, were found to be independently expressed in vitro. This was not the case, however, in vivo, where the upstream region appears to be required for maximal expression, a finding in accord with previous studies. Plasmids carrying a promoter deleted for this region displayed the same binding and rate characteristics as does p 2 in the wild type promoter. Thus, the intrinsic promoter strength is not affected by upstream sequences. I have also found ilvGp 2 to be highly dependent on negative supercoiling for maximal expression. Although the mechanism for this dependency remains unknown, it was found that sequences downstream of $-47$ were sufficient for specifying a promoter with characteristics identical to the wild type. Also, it was observed that p 1 and p 2 were differentially affected by supercoiling, resulting in a very significant change in promoter usage depending on the conformation of the template. I also studied pausing in the ilvG leader region and found that it could be enhanced in a non-competitive manner by ppGpp. In addition, deletion analysis from both 5$\prime$ and 3$\prime$ ends implicated both the 1:2 hairpin and a GC sequence downstream of this hairpin as being required for pausing.

Degree

Ph.D.

Subject Area

Biochemistry

COinS