Region 1.2 of the RNA polymerase σ subunit controls recognition of the −10 promoter element (original) (raw)
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Region 1.2 of the RNA polymerase sigma subunit controls recognition of the -10 promoter element
The EMBO journal, 2007
Recognition of the -10 promoter consensus element by region 2 of the bacterial RNA polymerase sigma subunit is a key step in transcription initiation. sigma 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 sigma70, whose function was heretofore unknown, is strictly required for efficient recognition of the non-template strand of -10-like pause-inducing DNA sequence by sigma region 2, and for sigma-dependent promoter-proximal pausing. Recognition of the fork-junction promoter DNA by RNA polymerase holoenzyme also requires sigma region 1.2 and thus resembles the pause-inducing sequence recognition. Our results, together with available structural data, support a model where sigma region 1.2 acts as a core RNA polymerase-dependent allosteric switch that modulates non-template DNA strand recognition by ...
The σ70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter
Nature Structural & Molecular Biology, 2004
The 70 subunit of RNA polymerase plays an essential role in transcription initiation. In addition, 70 has a critical regulatory role during transcription elongation at the bacteriophage late promoter, P R′ . At this promoter, 70 mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that 70 also mediates a pause in early elongation at the lac promoter (plac). Like that at P R′ , the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between 70 and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal 70 -dependent pauses may play a role in the regulation of many bacterial promoters.
The role of RNA polymerase subunit in promoter-independent initiation of transcription
Proceedings of the National Academy of Sciences, 2004
In bacteria, initiation of transcription depends on the RNA polymerase subunit, which brings catalytically proficient RNA polymerase core to promoters by binding to specific DNA elements located upstream of the transcription start point. Here, we study -dependent synthesis of a transcript that is used to prime replication of the single-stranded genome of bacteriophage M13. We show that, in this system, plays no role in DNA recognition, which is accomplished solely through RNA polymerase core interaction with DNA downstream of the transcription start point. However, is required for full-sized transcript synthesis by allowing RNA polymerase core to escape into productive elongation. RNA polymerase may play a similar role during replication primer synthesis in other bacterial mobile elements whose life cycle involves a single-stranded DNA stage.
Journal of Molecular Biology, 1998
The s subunit of RNA polymerase orchestrates basal transcription by ®rst binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli s 70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of s 70 to the closely related NarL or 434 Cro proteins, suggests that the ®ve basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the À35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a signi®cant effect on activator-independent transcription is at R603, indicating that this residue of s 70 may play a distinct role in transcription initiation.
Journal of Molecular Biology, 2011
Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable "open" complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RP o . The transcription initiation factor, σ 70 , plays critical roles in promoter recognition and RP o formation as well as in early steps of RNA synthesis.
Proceedings of the National Academy of Sciences, 2005
The -subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of and the flap domain of the RNAP -subunit (-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli 70 that have specific effects on the 70 region 4͞-flap interaction, either weakening or strengthening it. Using these 70 mutants, we demonstrate that the region 4͞-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the -flap, region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of -core interactions.
Cell, 2006
Regulation of transcription initiation is generally attributable to activator/repressor proteins that bind to specific DNA sequences. However, regulators can also achieve specificity by binding directly to RNA polymerase (RNAP) and exploiting the kinetic variation intrinsic to different RNAP-promoter complexes. We report here a previously unknown interaction with Escherichia coli RNAP that defines an additional recognition element in bacterial promoters. The strength of this sequence-specific interaction varies at different promoters and affects the lifetime of the complex with RNAP. Selection of rRNA promoter mutants forming long-lived complexes, kinetic analyses of duplex and bubble templates, dimethylsulfate footprinting, and zero-Angstrom crosslinking demonstrated that s subunit region 1.2 directly contacts the nontemplate strand base two positions downstream of the À10 element (within the ''discriminator'' region). By making a nonoptimal s1.2discriminator interaction, rRNA promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA, ultimately accounting for regulation of ribosome synthesis.
Biochemistry, 2012
Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free
Altered promoter recognition by mutant forms of the ?70 subunit of Escherichia coli RNA polymerase*1
J Mol Biol, 1989
We have systematically assayed the in viva promoter recognition properties of 13 mutations in rpoD, the gene that encodes the 0" subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of c" and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of cr", which is similar to the helix-turnhelix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the-35 region, and that a sequence internal to a", in a region which is highly conserved among Q factors, recognizes the-10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the-35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effects on promoter recognition.