Region 1.2 of the RNA polymerase σ subunit controls recognition of the −10 promoter element

Recognition of the −10 promoter consensus element by region 2 of the bacterial RNA polymerase σ subunit is a key step in transcription initiation. σ also functions as an elongation factor, inducing transcription pausing by interacting with transcribed DNA non-template strand sequences that are similar to the −10 element sequence. Here, we show that the region 1.2 of Escherichia coli σ(70), whose function was heretofore unknown, is strictly required for efficient recognition of the non-template strand of −10-like pause-inducing DNA sequence by σ region 2, and for σ-dependent promoter-proximal pausing. Recognition of the fork-junction promoter DNA by RNA polymerase holoenzyme also requires σ region 1.2 and thus resembles the pause-inducing sequence recognition. Our results, together with available structural data, support a model where σ region 1.2 acts as a core RNA polymerase-dependent allosteric switch that modulates non-template DNA strand recognition by σ region 2 during transcription initiation and elongation

Transcription reinitiation by recycling RNA polymerase that diffuses on DNA after releasing terminated RNA

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time. Bacterial transcription is terminated when RNA polymerases encounter terminator sequences. Using a single-molecule fluorescence assay, here the authors show that the release of transcript RNA precedes RNA polymerase dissociation and that the remaining RNA polymerase can reinitiate at nearby promoters

A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the β subunit and conserved region 4 of the σ subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the σ region 4/β-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the β-flap for binding to σ region 4, thereby inhibiting transcription initiation by disrupting the σ region 4/β-flap interaction