Generality of the Branched Pathway in Transcription Initiation byEscherichia coli RNA Polymerase

Abstract

Transcription initiation has been assumed to be a multi-step sequential process, although additional steps could exist. Initiation from the T7A1 promoter, in particular, apparently behaves in vitro in a manner that can be fully explained by the sequential pathway. However, initiation from the P R AL promoter has been shown to follow a branched pathway from which a part of the enzyme-promoter complex is arrested at the promoter raising the question as to which mechanism is general. We found that a moribund complex, characteristic of the arrested branch, is formed at the T7A1 promoter, especially in low salt condition indicating that the initiation mechanism for this promoter is also branched. The results of DNA footprinting suggested that holoenzyme in the moribund complex is dislocated on DNA from the position of productive complex. However, only a small fraction of the binary complex becomes arrested at this promoter, and the interconversion between subspecies of binary complex is apparently more reversible than at the P R AL promoter, which explains why the reaction pathway appears to be sequential. These findings suggest a generality of the branched pathway mechanism, which would resolve contradictory observations that have been reported for various promoters.Transcription initiation in prokaryotes includes at least four events: 1) the binding of holoenzyme to a promoter; 2) the isomerization of the resulting complex accompanied by strand opening; 3) the iterative synthesis and release of abortive transcripts; and 4) the achievement of continuous elongation accompanied by the escape of the enzyme from the promoter (for review see Ref. 1). Although these events are required for transcription initiation in this order, they do not necessarily represent the complete mechanism of transcription initiation, because additional steps could exist. Nevertheless, it has long been assumed that the events listed above is the complete mechanism, mainly because of the lack of evidence for further complications. This simplest mechanism may be called the sequential pathway (Scheme 1A). In vitro, the initiation from the bacteriophage T7A1 promoter in particular shows the following two behaviors that are characteristic of the sequential mechanism. Firstly, the promoter-RNA polymerase complex synthesizes a stoichiometric amount of full-length transcript in a single-round transcription (2). Secondly, abortive synthesis does not occur after the synthesis of full-length transcript (3), which is consistent with the view that the transcription complex engaged in abortive synthesis is a precursor of the complex synthesizing a long RNA. However, these results do not prove that the sequential pathway is applicable to initiation at all promoters or even at the T7A1 promoter in all conditions.The above two behaviors characteristic of the sequential pathway are not observed in single-round transcription from the P R AL or LacUV5 promoter. The amount of full-length transcript is much less than stoichiometric with the amount of the binary com...

Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription

“…For optimal promoters, RNAP binding generally results in an initial closed complex, which then quickly isomerizes to the open complex which is competent for transcription initiation (19,30,33). This branched pathway of RNAP-PcopA interactions is also distinct from the earlier branched pathway for transcription initiation at λP R AL and T7A1 promoters, where the branching occurs after forming the closed complex (37,38). Exploiting this branched interaction pathway, CueR biases the kinetic sampling of RNAP between the off-pathway RP DE and the RP O , leading to transcription repression or activation.…”

Ribosomal protein S1 promotes transcriptional cycling

“…These studies suggested a role of S1 beyond that in the ribosome. Here, we report the results of a detailed study of the effect of purified S1 on E. coli RNA polymerase transcriptional activity on DNA templates incorporating the extensively characterized bacteriophage T7A1 promoter (Dayton et al 1984;Reynolds et al 1992;Daube et al 1994;Susa et al 2002). Figure 1A shows a typical transcription-stimulatory effect resulting from the addition of purified native S1 to in vitro transcription reactions.…”

Tethering σ70 to RNA polymerase reveals high in vivo activity of σ factors and σ70-dependent pausing at promoter-distal locations

“…Some free or RNAP-bound could be sequestered nonspecifically. Promoter complexes also will sequester some s, but it is difficult to estimate how many because some may release slowly after initiation (Shimamoto et al 1986;Bar-Nahum and Nudler 2001;Mukhopadhyay et al 2001) and because some OCs appear unable to initiate transcription (Susa et al 2002). The extent to which these various interactions reduce 70 and RNAP availability depends on their avidity, but together they likely reduce substantially the effective concentrations of 70 and RNAP molecules.…”