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

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 alpha subunit (alpha CTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in alpha CTD severely impaired Spx-activated transcription. Attempts to substitute alanine for alpha 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 alpha 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 alpha D259, which has been implicated in alpha CTD-sigma(70) interaction (sigma(70) R603, corresponding to R362 of B. subtilis sigma(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 alpha CTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-alpha CTD complex. IMPORTANCE Though extensively studied in Escherichia coli, the role of alpha CTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal alpha CTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-alpha 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 alpha 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.