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 ternary elongation complex (TEC). Antiterminators modify the TEC so that it no longer responds to terminators. Together, terminators and antiterminators are used to control the level of gene expression. We study antiterminators that are unusual in that they are embedded in the nascent transcript. These antiterminators are encoded by the put sites of E. coli bacteriophage HK022. A nascent put transcript modifies the TEC that catalyzed its synthesis, thus decreasing transcription termination and increasing bacteriophage gene expression. Other TECs in the cell are unaffected. Put RNA dissociates from the TEC and terminators are no longer suppressed if the "tether" of nascent RNA that connects the put sequences to the active center is cleaved in vitro. Thus, antitermination requires persistent association of nascent put RNA with the TEC as it proceeds along the template, and the intrinsic affinity of the two components appears to be low. By contrast, we found that the integrity of the tether is not required for antitermination in vivo. In fact, the tether is efficiently cleaved by RNase III, a host endonuclease, just downstream of the put site. In spite of this, put RNA suppresses terminators located as far as 10 kbp from the put site in vivo. We offer possible explanations of the difference between the in vivo and in vitro results. First, put RNA dissociation from the TEC could be impeded in vivo by a "processivity" factor that is not present in vitro. Second, the rate of dissociation could be slow enough to allow the modification to persist for some time after the local concentration of put has decreased. To distinguish between these hypotheses, we will measure the rate of dissociation in vitro. We have shown that put suppresses the pausing that occurs when the TEC synthesizes transcripts that are rich in U residues. Such U-rich sequences are essential components of intrinsic terminators. When the TEC is artificially halted within a U-rich sequence, it moves backwards on the template with consequent disengagement of the 3' end of the transcript from the active site, and concerted retreat of the RNA:DNA hybrid region and its associated transcription bubble from the 3'-end. Such retrograde movement also exposes upstream phosphodiester bonds to hydrolysis activated by the GreB protein and delays resumption of elongation until the 3' end is restored to the active site by forward movement of the TEC. We found that put modification changed the properties of a TEC that is artificially halted at a U-rich sequence by a protein roadblock. A modified TEC was less sensitive to GreB-mediated cleavage, had an altered pattern of cleavage of the transcription bubble by KMnO4, and resumed transcription more rapidly upon removal of the roadblock than did an unmodified TEC. We propose that modification by put RNA favors the posttranslocational state of the TEC; that is, the state in which the catalytic site of the enzyme is engaged with the next incoming templated substrate. This activity of put could, in principle, account for its ability to suppress pausing and termination.