A secondary RNA polymerase sigma factor from Streptococcus pyogenes (original) (raw)
Related papers
F1000 - Post-publication peer review of the biomedical literature, 2011
Specific promoter recognition by bacterial RNA polymerase is mediated by p subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, p 70 (the housekeeping p subunit) and p S (an alternative p subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with p 70 (Ep 70) or with p S (Ep S) using the whole Escherichia coli genome as DNA template. We found that Ep 70 , in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, Ep S efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the À35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/ T content in the À10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the À10/+1 region could improve promoter recognition by Ep S .
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.
Nucleic Acids Research, 2011
Specific promoter recognition by bacterial RNA polymerase is mediated by p subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, p 70 (the housekeeping p subunit) and p S (an alternative p subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with p 70 (Ep 70) or with p S (Ep S) using the whole Escherichia coli genome as DNA template. We found that Ep 70 , in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, Ep S efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the À35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/ T content in the À10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the À10/+1 region could improve promoter recognition by Ep S .
Molecular Microbiology, 2009
The Pseudomonas sp. strain ADP protein AtzR is a LysR-type transcriptional regulator required for activation of the atzDEF operon in response to nitrogen limitation and cyanuric acid. Transcription of atzR is directed by the s N -dependent promoter PatzR, activated by NtrC and repressed by AtzR. Here we use in vivo and in vitro approaches to address the mechanisms of PatzR activation and repression. Activation by NtrC did not require any promoter sequences other than the s N recognition motif both in vivo and in vitro, suggesting that NtrC activates PatzR in an upstream activation sequences-independent fashion.
Role of the RNA polymerase sigma subunit in transcription initiation
Research in Microbiology, 2002
In bacteria, σ subunits direct the catalytically competent RNA polymerase core enzyme to promoters. Recent advances in our understanding of bacterial RNA polymerase reveal that σ subunits are intimately involved in all aspects of transcription initiation including promoter location, promoter melting, initiation of RNA synthesis, abortive initiation and promoter escape.
Methods in enzymology, 2003
The DNA-dependent RNA polymerase (RNAP; EC 2.7.7.6) of Escherichia coli, the best-characterized multisubunit RNAP, is composed of a core enzyme (E, subunit composition 2 0 !) and one of seven identified molecular species of the subunit (E, subunit composition 2 0 !). Advances in high-resolution structural analysis of the bacterial RNAP have opened up opportunities to study the functional role that each structural module of the RNAP plays in transcription. 1,2 A mobile structural module of E. coli RNAP, known as the downstream lobe (residues 186-433), was shown to contribute to stable open promoter complex formation during transcription directed by RNAP containing the 70 factor. 3 This article describes experimental systems used to probe the function of the subunit downstream lobe in the context of RNAP containing the major variant subunit, the enhancer-dependent factor, 54 . 4 Both enhancer-dependent RNAP ( 54 -RNAP) and enhancer-independent RNAP ( 70 -RNAP) are capable of promoter recognition that results in the formation of the closed promoter complex. 70 -RNAP closed promoter complexes can isomerize rapidly into transcriptionally active open promoter complexes in the absence of additional activators or energy sources. In contrast, 54 -RNAP complexes remain closed unless an enhancer DNA-bound activator and an energy source in the form of ATP or GTP hydrolysis is provided. The ATPase activity of the activator induces the propagation of initial DNA melting or distortion in the closed 54 -RNAP promoter complexes toward the transcription initiation start point and allows open promoter complex
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.
Activating Transcription in Bacteria
Annual Review of Microbiology, 2012
Bacteria use a variety of mechanisms to direct RNA polymerase to specific promoters in order to activate transcription in response to growth signals or environmental cues. Activation can be due to factors that interact at specific promoters, thereby increasing transcription directed by these promoters. We examine the range of architectures found at activator-dependent promoters and outline the mechanisms by which input from different factors is integrated. Alternatively, activation can be due to factors that interact with RNA polymerase and change its preferences for target promoters. We summarize the different mechanistic options for activation that are focused directly on RNA polymerase.
Science, 2002
plified by polymerase chain reaction (PCR) with primers containing 5Ј-homology arms corresponding to sequences flanking the regions to be deleted. Primers used to create LSR10 (⌬csgA) were 5Ј-gttaatttccattcgactttt-aaatcaatccgatgggggttttacGTGTAGGCTGGAGCTGCTTC and 5Ј-agggcttgcgccctgtttctgtaatacaaatgatgtAT TCCG-GGGATCCGTCGACC (lower-case letters correspond to csg sequences). The primers used to generate LSR5 (⌬csgDEFG;⌬csgBA), were 5Ј-agggcttgcgccctgtttctgtaa-tacaaatgatgtAT TCCGGGGATCCGTCGACC and 5Ј-gcc-gacatcaggcacagcataacaggttcgttcgagGTGTAGGCTGG-AGCTGCT TC. PCR products were electroporated into MC4100-expressing Red recombinase proteins from pKD46 (22). The resulting Kan r strains were confirmed by PCR and failed to bind CR when grown on YESCA plates. The mutation from LSR5 was transferred into C600 by standard P1 transduction, creating LSR6.