A two-subunit bacterial sigma-factor activates transcription in Bacillus subtilis

Abstract

The sigma-like factor YvrI and coregulator YvrHa activate transcription from a small set of conserved promoters in Bacillus subtilis. We report here that these two proteins independently contribute sigma-region 2 and sigma-region 4 functions to a holoenzyme-promoter DNA complex. YvrI binds RNA polymerase (RNAP) through a region 4 interaction with the beta-subunit flap domain and mediates specific promoter recognition but cannot initiate DNA melting at the -10 promoter element. Conversely, YvrHa possesses sequence similarity to a conserved core-binding motif in sigma-region 2 and binds to the N-terminal coiled-coil element in the RNAP beta'-subunit previously implicated in interaction with region 2 of sigma-factors. YvrHa plays an essential role in stabilizing the open complex and interacts specifically with the N-terminus of YvrI. Based on these results, we propose that YvrHa is situated in the transcription complex proximal to the -10 element of the promoter, whereas YvrI is responsible for -35 region recognition. This system presents an unusual example of a two-subunit bacterial sigma-factor.

Fig. 1.

YvrI region 4 interacts with the β-flap-tip helix. (A) Relevant amino acid sequence of E. coli and B. subtilis β-flap-tip regions (21). Two tip helix mutations (I905K and F906K) that impair in E. coli binding by σ⁷⁰-region 4 (21) are indicated (arrows). For interaction assays (BACTH), the region from amino acid 817 to 896 was fused to λcI. (B) BACTH analysis to test YvrI and YvrHa binding to B. subtilis β-flap. (C) BACTH analysis of the effect of I864K and F865K flap-tip mutations on the B. subtilis β-flap–YvrI region 4 interaction.

Fig. 2.

σ-Factor activities of YvrI. (A) YvrI competitively inhibits DNA melting by E. coli σ⁷⁰- and B. subtilis SigA. Reactions include E. coli σ⁷⁰-holoenzyme melting of the E. coli bla and B. subtilis ilv promoters and B. subtilis SigA holoenzyme melting of the ilv promoter. Competitor proteins [YvrHa, YvrI, and a control protein (BSA)] were either not added (control lane) or added at a concentration 5-fold and 10-fold over the concentration of holoenzyme (62 nM) before oxidation reaction using KMnO₄. (B) YvrI is a promoter specificity factor determined using EMSA and end-labeled YvrI-dependent oxdC promoter DNA (P_oxdC) and a control promoter DNA (P_yoeB). Each binding reaction contained the indicated concentration of RNAP (a) and 500 nM YvrHa (b), 500 nM YvrI (c), and 500 nM YvrHa and 500 nM YvrI (d). Note that the initial binding reaction in b, c, and d containing 0 nM RNAP includes the indicated amount of YvrI and/or YvrHa to control for nonspecific binding by these proteins.

Fig. 3.

σ-Factor region 2-like sequence in YvrHa includes functionally important residues. Composite alignments of the relevant sequences [regions 2.1, 2.2, and 2.3 as previously defined (4)] from a selection of primary and alternative σ-factors and the complete sequences of YvrHa and four YvrHa homologs. Highly conserved amino acids in regions 2.2 are highlighted in blue, whereas positionally conserved aromatic amino acids among the subgroups are highlighted in green. ClustalW was used to generate each individual alignment, and conservation symbols (asterisks) pertain to YvrHa homolog alignment only. For the alternative σ-factors: sequences SigM, SigX, SigW, SigY, and SigH are from B. subtilis; RpoE and RpoH are from E. coli; SigR is from Streptomyces coelicolor; and PvdS is from Pseudomonas aeruginosa. For the vegetative and YvrHa proteins, abbreviations are as follows: Bsu, B. subtilis; Eco, E. coli; Sco, S. coelicolor; Taq, Thermus aquaticus; Bth, Bacillus thuringiensis; Bcl, Bacillus clausii; Bce, Bacillus cereus; Bli, Bacillus licheniformis.

Fig. 4.

YvrHa interacts with the coiled-coil domain in β′-subunit. (A) BACTH analysis of YvrHa and YvrI interactions with β′ (amino acids 1–303). (B) Influence of mutations in the region 2.2-like motif (Fig. 3) of YvrHa on binding with β′ (1–303). (C) Sequence of the E. coli and B. subtilis coiled-coil element in β′ as previously defined (8). Mutations in E. coli β′ (R275Q, E294K, and A302D) that impair β′-binding to σ⁷⁰-region 2 are indicated with arrows. Approximate dimensions of helices that contribute to the coiled-coil domain are indicated by gray bars. (D) Influence of mutations in B. subtilis β′ (analogous to mutations shown in C) on β′-binding to YvrHa.

Fig. 5.

YvrI and YvrHa are required for promoter melting. Permanganate sensitivity of nucleotides in the template strand of P_oxdC as influenced by the addition of indicated proteins is shown. Nucleotide coordinates are shown in Fig. S6. Reactions contained B. subtilis RNAP (60 nM) with other proteins added to a final concentration of 300 nM.

Fig. 6.

YvrHa interacts with the amino terminus of YvrI. (A) BACTH analysis of interactions between YvrHa and either full-length YvrI or amino- and carboxyl-terminal segments of YvrI (Fig. S1). (B) In vitro pull-down assay using NHS-conjugated YvrHa or a control protein (lysozyme). Binding reactions included either full-length YvrI or the amino- or carboxy-terminal segment of YvrI. Arrows indicate bound amino-terminal fragment of YvrI. (C) In vitro pull-down assay testing interaction between immobilized YvrHa and either YvrI, SigA, or SigM. All test proteins were added to binding reactions to a final concentration of ≈1 μM.

Fig. 7.

Provisional model for a YvrI-YvrHa holoenzyme–promoter DNA closed transcription complex. Nonessential B. subtilis polymerase subunits (δ- and ω-subunits) are not included. The model depicts known interactions of YvrHa region 2-like segment (r2) with β′-coiled-coil domain (c-c), YvrHa with the amino terminus of YvrI, and YvrI region 4 (r4) with the β-subunit flap domain (flap). Drawing not to scale.