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The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor ?J modulates promoter recognition.

ABSTRACT: Extra-cytoplasmic function (ECF) ?-factors are widespread in bacteria, linking environmental stimuli with changes in gene expression. These transcription factors span several phylogenetically distinct groups and are remarkably diverse in their activation and regulatory mechanisms. Here, we describe the structural and biochemical features of a Mycobacterium tuberculosis ECF factor ?J that suggests that the SnoaL_2 domain at the C-terminus can modulate the activity of this initiation factor in the absence of a cognate regulatory anti-? factor. M. tuberculosis ?J can bind promoter DNA in vitro; this interaction is substantially impaired by the removal of the SnoaL_2 domain. This finding is consistent with assays to evaluate ?J-mediated gene expression. Structural similarity of the SnoaL_2 domain with epoxide hydrolases also suggests a novel functional role for this domain. The conserved sequence features between M. tuberculosis ?J and other members of the ECF41 family of ?-factors suggest that the regulatory mechanism involving the C-terminal SnoaL_2 domain is likely to be retained in this family of proteins. These studies suggest that the ECF41 family of ?-factors incorporate features of both-the ?70 family and bacterial one-component systems thereby providing a direct mechanism to implement environment-mediated transcription changes.

SUBMITTER: Goutam K

PROVIDER: S-EPMC5766207 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition.

Goutam Kapil K Gupta Arvind K AK Gopal Balasubramanian B

Nucleic acids research 20170901 16

Extra-cytoplasmic function (ECF) σ-factors are widespread in bacteria, linking environmental stimuli with changes in gene expression. These transcription factors span several phylogenetically distinct groups and are remarkably diverse in their activation and regulatory mechanisms. Here, we describe the structural and biochemical features of a Mycobacterium tuberculosis ECF factor σJ that suggests that the SnoaL_2 domain at the C-terminus can modulate the activity of this initiation factor in the ...[more]

PMID: 28934483

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Project description:To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpress sigB and sigF. Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes after sigB induction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression of sigB and sigF. Overexpression of sigB resulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction of sigB did not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of the M. tuberculosis sigma factor regulatory cascade. Analysis of the 5'-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N(14-18)-NNGNNG. This sequence appeared upstream of both sigB (Rv2710) and the gene following it, ideR (Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression of sigF revealed that only the sigC gene was significantly upregulated 6 and 12 h after sigF induction. The previously identified SigF promoter consensus sequence AGTTTG-N(15)-GGGTTT was identified in the 5' UTR of the sigC gene, and SigF-dependent in vitro transcription of the promoter upstream of sigC was confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression of sigB and sigF resulted in reduced rates of M. tuberculosis intracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

Dataset Information

The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor ?J modulates promoter recognition.

Publications

The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition.

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