9760–9772 Nucleic Acids Research, 2017, Vol. 45, No. 16 Published online 18 July 2017 doi: 10.1093/nar/gkx609 The fused SnoaL 2 domain in the Mycobacterium tuberculosis sigma factor ␴J modulates promoter recognition Kapil Goutam, Arvind K. Gupta and Balasubramanian Gopal* Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, Karnataka, India Received April 11, 2017; Revised July 01, 2017; Editorial Decision July 04, 2017; Accepted July 06, 2017 ABSTRACT acterized ␴70 members rely on protein–protein interactions to enable DNA promoter binding––a key distinction from Extra-cytoplasmic function (ECF) ␴-factors are the ␴54 class of ␴ factors that can form tight DNA com- widespread in bacteria, linking environmental stim- plexes but require adenosine triphosphate hydrolysis for uli with changes in gene expression. These tran- open promoter complex formation (2,3). ␴70 members have scription factors span several phylogenetically dis- been further classified into groups based on domain archi- tinct groups and are remarkably diverse in their ac- tecture (4,5). While the housekeeping ␴ factor contains an tivation and regulatory mechanisms. Here, we de- N-terminal polypeptide segment (region 1.1) and four DNA scribe the structural and biochemical features of a binding domains and governs basal expression of genes, Mycobacterium tuberculosis ECF factor ␴J that sug- the extra-cytoplasmic function (ECF) ␴ factors are much gests that the SnoaL 2 domain at the C-terminus can smaller with only two DNA binding domains (referred to as ␴ ␴ modulate the activity of this initiation factor in the 2 and 4) and govern transcription under stress or starva- ␴ absence of a cognate regulatory anti-␴ factor. M. tu- tion conditions (6). ECF factors are the largest and most divergent group in ␴70 ␴ factors and govern transcription in berculosis ␴J can bind promoter DNA in vitro;this response to various stresses and starvation conditions. The interaction is substantially impaired by the removal activity of most ECF ␴ factors (and a few members of other of the SnoaL 2 domain. This finding is consistent ␴ factor families) is regulated by interaction with a protein J with assays to evaluate ␴ -mediated gene expres- antagonist also referred to as an anti-␴ factor (7). The anti- sion. Structural similarity of the SnoaL 2 domain with ␴ factor can be either cytoplasmic or membrane bound. epoxide hydrolases also suggests a novel functional The release of a ␴ factor from these ␴/anti-␴ complexes role for this domain. The conserved sequence fea- is brought about by diverse mechanisms including pro- tures between M. tuberculosis ␴J and other mem- teolysis, phosphorylation and redox-dependent conforma- bers of the ECF41 family of ␴-factors suggest that tional changes (8–10). More recently, ECF group members the regulatory mechanism involving the C-terminal were classified into 43 sub-groups based on sequence ar- SnoaL 2 domain is likely to be retained in this family chitecture (11). Another study utilizing under-represented genomes extended these subgroups to more than 50 (12). of proteins. These studies suggest that the ECF41 ␴ Four of the ECF groups viz ECF41, ECF42, ECF44 and family of -factors incorporate features of both––the ECF01-Gob contain an additional domain at the carboxyl- ␴70 family and bacterial one––component systems terminus. Of these, the ECF44 sub-group ␴ factors which thereby providing a direct mechanism to implement contain a conserved carboxyl-terminal cysteine rich domain environment-mediated transcription changes. (CRD) have been better characterized (13–15). The activity of two ECF44 ␴ factors, Myxococcus xanthus corE1 and corE2 is directly regulated by metal ions. While corE1 re- INTRODUCTION sponds to Cu, corE2 binds Cd and Zn and mutation of key Regulation of gene expression in prokaryotes occurs pri- cysteine residues in the CRD affects metal ion binding. In- marily at the transcription initiation step. ␴ factors, the deed, deletion of the ECF44-specific Cysteine-X-Cysteine ␴ specificity defining subunit of RNA polymerase (RNAP), (CXC) motif from 2 results in loss of activity (14,15). govern gene expression by their reversible association with The ECF41 ␴ factor sub-group, that contains nearly the RNAP (1). Of the two broad families of ␴ factors, mem- 400 annotated members distributed across 10 phyla, is bers of the the ␴70 family are more diverse due to variations poorly understood. All ECF41 group members possess a ␴ ␴ in their activation and regulatory mechanisms. Most char- distinct domain organization with 2, 4 and an additional *To whom correspondence should be addressed. Tel: +91 80 2293 3219; Fax: +91 80 2360 0535; Email: [email protected] C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2017, Vol. 45, No. 16 9761 carboxy-terminal domain (11,16). ECF41 members lack an MATERIALS AND METHODS apparent anti-␴ factor and the regulation of ECF41 ␴ fac- Expression and purification of recombinant proteins tor activity remains unclear. In the absence of an antagonist (anti-␴ factor), the domain at the C-terminal has been sug- The details of expression constructs used to express and pu- gested to play a role similar to an anti-␴ factor. A recent rify ␴J (Rv3328c) are summarized in Supplementary Ta- study of two ECF41 group ␴ factors from Bacillus licheni- ble S1. All the proteins used in crystallization and inter- formis and Rhodobacter sphaeroides suggested a regulatory action assays were purified using the same protocol unless role for this additional domain at the Carboxy-terminus. otherwise mentioned. Clones were confirmed using single These studies, performed using deletion analysis and inter- primer based sequencing (Amnion Biosciences Pvt. Ltd.). action assays, proposed the additional domain to be a fused In each case, the plasmid with the gene of interest was trans- anti-␴-factor-like domain with a potential role in promoter formed into Escherichia coli BL21(DE3) strain (Novagen activation as well as interaction with the RNAP. (16). An Inc.). A single colony was inoculated in Luria–Bertani interesting observation is that of genomic context––ECF41 medium or minimal medium (for seleno-methionine (Se- genes are often next to those of carboxy muconolactone de- Met) derivative) containing an appropriate antibiotic. Cul- carboxylases, oxidoreductases or epimerases (referred to as tureswereallowedtogrowuptoOD600nm of 0.5–0.6 at COE) (11,16). This genomic proximity suggested a possible 37◦C prior to induction with 0.2 mM Isopropyl ␤-D-1- role for this ␴ factor in maintaining redox homeostasis. thiogalactopyranoside (IPTG). For the Se-Met derivative, Mycobacterium tuberculosis ␴ factors play a critical role amino acid supplements and Se-Met were added at an in the virulence of this human pathogen (17). M. tubercu- OD600nm of 0.4. Post-induction, cultures were grown for losis has two ECF41 ␴ factors ␴I and ␴J (11). The cellular 12 h at 18◦C. Cells were then spun at 7000 g for 15 min. level of ␴J is upregulated in late stationary phase (18). The The pellet was re-suspended in buffer A (50 mM Tris–HCl only known gene under M. tuberculosis ␴J regulation is that pH8.0, 300 mM NaCl) containing 2 mM phenylmethane- encoding ␴I (19). Although the M. tuberculosis sigJ gene is sulfonyl fluoride (PMSF) and ethylenediaminetetraacetic not flanked by any of the COE genes, the target sigI gene has acid (EDTA)-free protease inhibitor tablets (Roche). Cells a putative proline dehydrogenase in the proximity. Interest- were lysed by sonication and the cell debris were removed ingly, M. tuberculosis ECF41 ␴ factor ␴J has been reported by centrifugation at 30 000 g for 45 min at 4◦C. The to influence resistance to hydrogen peroxide-mediated ox- supernatant was incubated with Ni-NTA resin (Sigma– idative stress (20). Another study on M. marinum ␴ factors Aldrich) for 1 h. The protein was then eluted using buffer under different stress conditions suggests the possible in- A with a gradient of imidazole (10–200 mM). The pro- volvement of ␴J in heat stress (21). The lack of an appar- tein was further purified by size-exclusion chromatography ent anti-␴ factor that is a receptor for redox stimuli made using Sephacryl S200 Hi-Prep 16/60 column (GE Health- it interesting to explore whether the additional domain in care, Inc.). The fractions containing the purified protein ␴J plays a role akin to an anti-␴ factor. The C-terminal do- were concentrated to ca 10 mg/ml for crystallization trials. main in ␴J was predicted to be a SnoaL 2 domain that is The purity and molecular mass of the protein was further classified under the nuclear transport factor 2 (NTF2-like) verified using sodium dodecyl sulphate-polyacrylamide gel superfamily (22). These proteins do not contain any specific electrophoresis (SDS-PAGE) and liquid chromatography– conserved sequence motifs but share a common structural electrospray ionization mass spectrometry (LCESI-MS) fold (23,24). The NTF-2 like fold is a cone shaped struc- (Bruker Daltonics, Inc.). Se-Met labeled protein was pu- ture with an internal cavity. Proteins with this fold are in- rified similarly with the exception that 1 mM Tris-(2- car- volved in a wide range of functions––both enzymatic and boxyethyl)phosphine or 2 mM dithiothreitol (DTT) was in- non-enzymatic. The catalytically active members include cluded during the purification. SnoaL polyketide cyclases, limonene-1,2-epoxide hydrolase and delta-5-3-ketosteroid isomerases (25–28). In the case Crystal structure determination of a characterized non-catalytic role, the NTF2 domain of ␴J the calcium/calmodulin-dependent protein kinase II is in- Crystallization trials for have been described earlier (30).