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

Abstract

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 absence of a cognate regulatory anti-σ factor. M. tuberculosis σJ 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 σJ-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 σJ and other members of the ECF41 family of σ-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 σ-factors incorporate features of both—the σ70 family and bacterial one—component systems thereby providing a direct mechanism to implement environment-mediated transcription changes.

The role of C‐terminal extensions in controlling ECF σ factor activity in the widely conserved groups ECF41 and ECF42

“…Strikingly, when mapping these co‐varying residues to the three‐dimensional structure of SigJ (PDB: 5XE7 (Goutam et al , )), we found that most of these top‐scoring predictions lie in close three‐dimensional proximity – except for the pair in σ 2 and the N‐terminus of the extension (Fig. A–C) – supporting the idea that the predicted interactions may be in physical contact.…”

“…Despite first insights into the phenomenology of σ factor control via C‐terminal extensions, the molecular mechanisms that govern this complex regulation are largely unknown. A recently described crystal structure of SigJ from Mycobacterium tuberculosis revealed that the SnoaL‐like extension might serve as a scaffold for the σ 2 and σ 4 domains in ECF41 (Goutam et al , ), potentially sequestering the σ factor into an inactive conformation. But so far, there is no structural information available for ECF groups containing other C‐terminal extensions that could help elucidating the details of their regulatory mechanism.…”

“…Here, we apply DCA to groups ECF41 and ECF42 with the aim of better understanding the regulatory role of their C‐terminal extensions. As a proof of concept, we first focused on ECF41 and benchmarked the contact predictions of DCA against the crystal structure of SigJ from M. tuberculosis (Goutam et al , ). We found that the regions with the highest co‐variation scores reside in close proximity in the structure of SigJ and verified experimentally that these contacts are indeed important for the inactivation of Ecf41 Bli from Bacillus licheniformis .…”

Characterization of an ECF56-family sigma factor fromStreptomyces venezuelaereveals a highly conserved regulome

“…Initial studies in the ECF41 subfamily in B. lincheniformis (Firmicutes) and R. sphaeroides (α-proteobacteria) found that truncations of the NTF2 domain drastically upregulated transcription of a highly conserved promoter (12). Interestingly, biochemical and crystal structure studies of the ECF41 σ J of M. tuberculosis ( Actinobacteria ) and the ECF42 of S. venezuelae ( Actinobacteria ) revealed that the NTF2 domain was necessary for transcription (9, 10). While a direct coupling analysis of ECF41 and ECF42 revealed that the NTF2 region bind and regulate the core ECF regions of σ 2 and σ 4 responsible for transcription (11), the role of these NTF2 domains in the ECF56 subfamily has not yet been studied.…”

“…The NTF2 superfamily of domains is widely distributed on the tree of life and shows little conservation at the primary sequence identity level; but members include many enzymes and a recent review of their function has implicated allosteric regulation of DNA binding proteins (8). Studies of these NTF2 domains in ECF41 and ECF42 found it was critical to initiation of gene transcription and strongly implicate that it directly responds to an external signal (9–12). In 2015, a genomic survey of ECFs and other signal transduction proteins in Actinobacteria revealed another group of ECFs with similar C-terminal extensions: the ECF56 subgroup (13).…”