Our objective is to understand how RNA polymerase of E. coli is modified by the transcription antiterminator encoded by the phage lambda gene Q. Antitermination is an important mechanism of genetic regulation in bacteria, as well as eukaryotes - for example in the growth of HIV. Furthermore, expression of many cellular genes may be controlled by related processes that act at the level of transcript elongation. The extensive conservation of the large subunits of all RNA polymerases, which are mostly responsible for enzyme movement and RNA chain growth, suggests that there will be important common features in mechanisms of elongation control in many organisms. Interaction of E. coli RNA polymerase with the single 23 kD Q polypeptide, along with the accessory protein NusA, changes the elongation properties of the enzyme so that it goes through transcription terminators, and is less susceptible to some signals that induce pausing. Q modifies RNA polymerase at a well defined site that includes the late gene promoter, a Q binding site in DNA, and a DNA signal that induces transcriptional pausing; this signal acts primarily through interactions with the hon-transcribed strand. The action of Q on enzyme paused at this site may simulate Q function at terminators, because it promotes elongation through the pause. We will characterize this interaction and determine its relation to the activity of Q protein at terminators, and characterize the process of termination itself. We will dissect the Q protein to identify portions involved in DNA binding and in its interaction with RNA polymerase, using both genetic and biochemical methods. We will determine how the modification induced by Q persists from the promoter-associated engagement site throughout the process of transcription elongation.
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