Control Mechanisms In Temperate Bacteriophage

Child Health And Human Development

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Abstract

Transcription terminators stop elongation of transcripts by DNA-dependent RNA polymerase and dissociate the transcript elongation complex. Antiterminators modify the elongation complex so that it no longer responds to terminators. Together, they are used to control gene expression. We study antiterminators that are unusual in that they are embedded in the nascent transcript. These antiterminators, which are encoded by the put sites of E. coli bacteriophage HK022, bind to and modify the elongation complexes that catalyzed their synthesis. This suppresses transcription termination and increases gene expression. We have recently shown that the chemical stability of put RNA is considerably greater than that of the typical E. coli message because the elongation complex protects this RNA from degradation. As far as we know, this is the first example of transcript stabilization mediated by binding to the elongation complex. RNA stability decreased more than 50-fold when binding to the elongation complex was prevented by mutation. The functional modification conferred by put RNA on the elongation complex is also long-lived: the efficiency of terminator suppression remained high for up to 10 kbp from the putL site. In addition, a cellular ribonuclease rapidly and efficiently cleaved the transcript just downstream of the put sequences, but such cleavage changed neither the stability of put RNA nor the efficiency of antitermination. Thus, the continuity of the RNA that connects put sequences to the growing point (the "tether") is not required for persistence of the antiterminating modification in vivo. These results led us to reinvestigate earlier findings indicating that binding of put RNA to the elongation complex is destabilized by cleavage of the tether in vitro. We now find that at least a fraction of put RNA remains bound to the elongation complex in a stable, salt-resistant complex after the tether is cleaved. We are currently studying the antitermination properties of these stable complexes and plan to characterize the binding surfaces.[unreadable] [unreadable] We have shown that put suppresses transcriptional pausing at a U-rich sequence located immediately downstream of the put site. This pause is associated with backtracking of the elongation complex, and we have shown that backtracking is inhibited by binding of put RNA to the elongation complex. However, when the U-rich sequence was moved to a more distant location, pausing was no longer suppressed. We conclude that suppression of pausing at the original location results from steric occlusion of the backtracked region by association of nascent put RNA with the elongation complex, and is not a consequence of persistent modification of the elongation properties of RNA polymerase. This argues against the hypothesis that put suppresses termination by favoring continued elongation at U-rich sequences.[unreadable] [unreadable] We have completely sequenced and are now annotating the genome of a bacteriophage, B40-8, whose host is Bacteroides fragilis, a human commensal bacterium that is frequently pathogenic. The sequence shows that this virus is a very distant relative of all known bacterial viruses. Indeed, about three-quarters of the open reading frames have no known homologs. We now plan to determine the transcription pattern of B40-8, with the aim of identifying regulatory factors and sites. In view of the dissimilarity of B40-8 to known organisms, we expect that this will reveal hitherto unknown mechanisms of transcriptional control. We will also determine the function of some of the unknown open reading frames and develop B40-8 as a transducing phage for B. fragilis.