Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis

Abstract

Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP ␣ subunit (␣CTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in ␣CTD severely impaired Spx-activated transcription. Attempts to substitute alanine for ␣CTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoA Y263C mutant, where "Y263C" indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound ␣CTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoA E255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli ␣D259, which has been implicated in ␣CTD-70 interaction ( 70 R603, corresponding to R362 of B. subtilis A ). However, the combined rpoA E255A and sigA R362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while ␣CTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-␣CTD complex.IMPORTANCE Though extensively studied in Escherichia coli, the role of ␣CTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal ␣CTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-␣CTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several ␣CTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.KEYWORDS alpha subunit, RNA polymerase, Spx, transcription, sporulation R egulation of gene expression at the level of transcription initiation is required for bacteria to maintain fitness and survival in a dynamic environment (e.g., perturbations in temperature, nutrient availability, exposure to toxic agents, etc.). Global control

Spx, the central regulator of the heat and oxidative stress response in B. subtilis, can repress transcription of translation‐related genes

“…As Spx binds close to the surface of α‐CTD, which contacts the upstream UP elements (Gaal et al , ; Zhang et al , ; Birch et al , ), it could act by influencing the recognition and productive interactions of α‐CTD with the specific upstream sequences. The strong transcription of rRNA promoters is also dependent on these UP‐elements (Fig.…”

“…In addition, it was recently observed that Spx interacts not only with α but also β or β′ subunits of RNAP (Birch et al , ). Such a more complex interaction or different binding mode of Spx and RNAP might also contribute to the inhibition of transcription at the rrnJ P1 promoter.…”

Engineering of Bacillus Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit

“…Compared with previously reported UP elements in P trxA (terms as UPA here), UPL contained a high-consensus sequence of “AAGAAATAAT” and sheared adenine (A) and thymine (T) dinucleotide repeat tracts. This has been proven to be a crucial element for RNAP recognition and binding in B. subtilis [ 29 ]. However, there were significant differences between the two UP elements in terms of genomic location.…”

Sense and sensor ability: redox-responsive regulators in Listeria monocytogenes

“…Oxidative stress also induces an intramolecular disulfide bond in the Spx CxxC redox switch, altering its conformation such that it interacts with RNA polymerase to regulate downstream genes (Fig. 2A) [25,26]. In B. subtilis, oxidized Spx activates over 100 genes involved in maintaining redox homeostasis (e.g.…”