Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

Abstract

Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP ␣ subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at ؊44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP A subunit from a ؊35-like element upstream of the trxB (thioredoxin reductase) promoter to positions ؊36 and ؊11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP. Bacterial responses to environmental and metabolic changes often involve gene regulation at the level of transcription initiation. The essential enzyme catalyzing this first step of gene expression, RNA polymerase (RNAP), is targeted by a variety of regulatory factors that direct RNAP activity to specific transcription units (1). The core RNAP, bearing the catalytic component, is composed of large subunits, ␤ and ␤=, the ␣ subunit dimer, and the subunit. Interaction of core RNAP with the subunit gives rise to the holoenzyme endowed with gene promoter specificity (2, 3). Regulatory factors that are controlled by signal sensing/ transducing systems that mediate stress responses can target one or more of the RNAP subunits to modulate activity and promoter specificity. Some regulatory factors exert positive control by engaging promoter DNA and recruiting RNAP through direct protein-RNAP subunit interaction (1). There is a growing list of regulatory proteins that prerecruit or appropriate RNAP by first contacting holoenzyme before mediating DNA target recognition (4). One of them, AsiA of phage T4, serves to mediate contact between RNAP subunit and a phage-encoded DNA-bound factor, MotA, which triggers prereplicative phage gene transcription (5). The DksA/Gre family proteins target the RNAP active site to affect transcription initiation and elongation at specific promoters (6). SoxS and related proteins engage in prerecruitment by interacting with holoenzyme before contacting specific cis-acting promoter elements (7). Other factors, such as Crl and 6S RNA, stabilize or inhibit specific holoenzyme forms beari...

Transcription of Oxidative Stress Genes Is Directly Activated by SpxA1 and, to a Lesser Extent, by SpxA2 in Streptococcus mutans

“…For example, mutational analysis suggests that the R92 residue is also important for redox control (24,41). While the S. mutans SpxA1 possesses the R92 residue, the arginine is replaced by a serine (S92) in SpxA2.…”

“…Representatives of three or more independent reactions are shown. 41). In a separate study, standard computational searches for consensus Spx-RNAP binding motifs in B. subtilis were unsuccessful (36).…”

Streptococcus mutans NADH Oxidase Lies at the Intersection of Overlapping Regulons Controlled by Oxygen and NAD + Levels

“…The Spx regulator senses the redox state through a CXXC motif and has been described previously in many firmicutes, including Bacillus subtilis (33)(34)(35)(36). Two homologs of Spx, SpxA (SMU.1142) and SpxB (SMU.2084), have been described in S. mutans (32,37).…”

Evidence that Oxidative Stress Induces spxA2 Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation

“…The promoter region of the major transcriptional start site of spxA2 resembles those recognized by the A form of RNAP, with the start site of transcription located 223 bp from the ATG start codon (41). Previous studies of Spx in B. subtilis uncovered the importance of an AGCA motif centered at approximately position Ϫ44 of Spx-controlled promoters as a possible cis-acting element (30,31), which was supported by genomewide Spx-DNA binding studies (10) and by nucleotide-specific protein-DNA cross-linking studies (37). The spxA2 promoter region does not possess the AGCA element in the vicinity of the Ϫ35 core promoter element.…”

“…However, mutational analysis of Spx-controlled promoter DNA and nucleotide-specific protein-DNA cross-linking provided evidence that RNAP-bound Spx contacts a conserved cisacting element in B. subtilis (10,30,31,37). The Spx-targeted cis-acting site, AGCA centered around position Ϫ44 upstream of the transcription start site, was detected in genome-wide chromatin immunoprecipitation studies (10).…”