Mutually Exclusive Utilization of P _R and P _RM Promoters in Bacteriophage 434 O _R

Abstract

Establishment and maintenance of a lysogen of the lambdoid bacteriophage 434 require that the 434 repressor both activate transcription from the P RM promoter and repress transcription from the divergent P R promoter. Several lines of evidence indicate that the 434 repressor activates initiation of P RM transcription by occupying a binding site adjacent to the P RM promoter and directly contacting RNA polymerase. The overlapping architecture of the P RM and P R promoters suggests that an RNA polymerase bound at P R may repress P RM transcription initiation. Hence, part of the stimulatory effect of the 434 repressor may be relief of interference between RNA polymerase binding to the P RM promoter and to the P R promoter. Consistent with this proposal, we show that the repressor cannot activate P RM transcription if RNA polymerase binds at P R prior to addition of the 434 repressor. However, unlike the findings with the related phage, formation of RNA polymerase promoter complexes at P RM and at P R apparently are mutually exclusive. We find that the RNA polymerase-mediated inhibition of repressor-stimulated P RM transcription requires the presence of an open complex at P R . Taken together, these results indicate that establishment of an open complex at P R directly prevents formation of an RNA polymerase-P RM complex.Each lambdoid bacteriophage contains a right operator (O R ) region on its chromosome that is at the center of a complex regulatory circuit responsible for governing the phage's choice between lytic and lysogenic development. Proper regulation of transcription initiation from the divergently oriented P R and P RM promoters that lie within the O R region is crucial to the lysis-lysogeny decision. In each phage, the activities of these promoters are regulated, in part, by the binding of the bacteriophage repressor to three recognition sites that partially overlap the P R and P RM promoters. In the absence of the repressor, the P RM promoter is virtually inactive and RNA polymerase preferentially initiates transcription at the P R promoter. During an infection or induction of a lysogen, continued activity of the P R promoter drives the phage to develop lytically. If the phage is to develop or maintain the lysogenic state, there must be exclusive expression of P RM over P R .To perform its role in the lysis-lysogeny decision, the repressor must bind to each of the three binding sites or operators within O R with different affinities and act both as transcriptional activator of P RM and as repressor of P R . In a developing or existing lysogen, the repressor binds with highest affinity to two sites, O R 1 and O R 2. In this configuration, the repressor molecule bound at O R 2 activates transcription from the P RM promoter. This event leads to expression of the cI gene that encodes the repressor, which is the sole protein responsible for maintenance of the lysogenic state. This binding configuration also permits the repressor to concurrently inhibit transcription initiation from P R and in doing so preven...

Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage

“…The relative positioning of repressor binding sites with respect to the P R promoter elements appears to determine the mechanism the phage repressor uses to repress P R transcription (51). In bacteriophage 434, the Ϫ35 and Ϫ10 regions of the P R promoter surround, but do not overlap, the O R 2 site (51).…”

MarA-mediated Transcriptional Repression of the rob Promoter

“…Because helical dependence is a critical factor in activation by MarA (19), the transcriptional outcome, repression versus activation, may be dependent on which DNA face the protein binds as is the case for GalR (41). The phage 434 protein represses the P r promoter via a binding site that lies between the Ϫ10 and Ϫ35 hexamers (33,42). Ethylation interference experiments at this promoter demonstrate that repression occurs via the binding of the 434 repressor and RNAP on opposite faces of the promoter (33).…”

Transcriptional interference – a crash course

“…Examples of TI as a means of gene regulation are common in the genomes of 'extrachromosomal' elements such as bacteriophages [19,[36][37][38], transposable elements [39], insertion sequences [40] and plasmids [25,26]. Perhaps the highly compact nature of these genomes favours the evolution of convergent transcription to regulate gene expression.…”