Interplay between σ region 3.2 and secondary channel factors during promoter escape by bacterial RNA polymerase

Abstract

In bacterial RNA polymerase (RNAP), conserved region 3.2 of the σ subunit was proposed to contribute to promoter escape by interacting with the 5'-end of nascent RNA, thus facilitating σ dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled σ subunit of RNAP. We show that substitutions and deletions in σ region 3.2 decrease the rate of promoter escape and lead to accumulation of inactive complexes during transcription initiation. Secondary channel factors differentially regulate this process depending on the promoter and mutations in σ region 3.2. GreA generally increase the rate of promoter escape; DksA also stimulates promoter escape on certain templates, while GreB either stimulates or inhibits this process depending on the template. When observed, the stimulation of promoter escape correlates with the accumulation of stressed transcription complexes with scrunched DNA, while changes in the RNA 5'-end structure modulate promoter clearance. Thus, the initiation-to-elongation transition is controlled by a complex interplay between RNAP-binding protein factors and the growing RNA chain.

DNA template sequence control of bacterial RNA polymerase escape from the promoter

“…The data in Figure 3B and D show that fraction of open complexes that were unable to escape was ∼15–30% and no clear pattern of dependence on ITS could be detected. In the presence of GreB a modest reduction of the fraction of complexes that did not escape could be observed ( Figure S2C, Supplementary Information ), in agreement with previously proposed role of backtracking in the formation of non-productive initiation complexes ( 31 ) and recent kinetic investigation ( 46 ). Decrease of open complex stability ( 47 ) could also contribute to the observed effect of GreB on fraction of complexes that did not escape.…”

“…The fact that slow kinetic component was not completely eliminated by GreB ( Figure S2A and B, Supplementary Information ) may indicate presence of additional mechanisms for delaying escape that do not respond to GreB. Recent report on promoter escape kinetics ( 46 ) that used real-time escape assay utilizing fluorescence signal of a probe incorporated into σ subunit also found that generally GreA and GreB increased the rate of escape but on some templates GreB could also inhibit escape. While the observed effects of GreB on escape rates are consistent with the presence of significant amounts backtracking complexes during escape under our experimental conditions, a detailed study of GreA/GreB effects on escape (and template-sequence dependence of these effects) will be needed to fully understand these effects.…”

Structural basis for transcription initiation by bacterial ECF σ factors

“…The structure predicts that the σ A 3.2 finger has to be displaced by an RNA molecule of > 4nt length and that the σ A 3.2 loop in the RNA exit channel has to be cleared during the promoter escape process. The interactions observed in the σ A -RPo structure underscore the key role of the σ A 3.2 on abortive production, pausing, and promoter escape in transcription initiation 28,29,49,50 . In our crystal structures of σ H -RPo, we show that σ H 3.2 guides the template ssDNA into the active site cleft and forms interactions with template ssDNA ( Figure 3D and 5E).…”

“…Domain σ3.2 reaches into the RNAP active site cleft and "pre-organizes" template ssDNA 24 . Domain σ3.2 also blocks the path of the extending RNA chain (> 5nt) 26,27 thereby contributing to both initial transcription pausing 28 and promoter escape 29,30 .Each category of known ECF σ factors recognizes promoters bearing a unique sequence signature at the -35 and the -10 elements 10,31 . In contrast to the high tolerance to sequence variation at the -35 and the -10 promoter elements exhibited by the primary σ factor, the ECF σ factors have stringent requirements for sequence identity in the -35 and the -10 elements and for spacer length between these two elements through an unknown mechanism 8, 32 .Although both the primary and ECF σ factors recognize the -35 element via σ4 and recognize the -10 element via σ2, the protein sequences of these two domains are not well conserved, and the consensus sequences of the two corresponding DNA elements vary.…”

“…Domain σ3.2 reaches into the RNAP active site cleft and "pre-organizes" template ssDNA 24 . Domain σ3.2 also blocks the path of the extending RNA chain (> 5nt) 26,27 thereby contributing to both initial transcription pausing 28 and promoter escape 29,30 .…”