Birch Cierra A, Davis Madison J, Mbengi Lea, Zuber Peter
Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health and Science University, Portland, Oregon, USA.
Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health and Science University, Portland, Oregon, USA
J Bacteriol. 2017 Jun 27;199(14). doi: 10.1128/JB.00124-17. Print 2017 Jul 15.
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 -encoded RNAP α subunit (αCTD). Previous mutational analysis of revealed that substitutions of Y263 in αCTD severely impaired Spx-activated transcription. Attempts to substitute alanine for αCTD 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 mutant alleles in the mutant, where "" indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound αCTD amino acid residues are not essential for Spx-activated transcription but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to αD259, which has been implicated in αCTD-σ interaction (σ R603, corresponding to R362 of σ). However, the combined and mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while αCTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-αCTD complex. Though extensively studied in , the role of αCTD in activator-stimulated transcription is largely uncharacterized in Here, we conduct phenotypic analyses of putatively lethal αCTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-αCTD-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 αCTD 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.
Spx是一种在革兰氏阳性菌中保守的全局转录调节因子，在革兰氏阳性菌中，Spx对于预防氧化诱导的蛋白毒性是必需的。在应激诱导时，Spx通过与编码的RNA聚合酶α亚基（αCTD）的C末端结构域相互作用来结合RNA聚合酶（RNAP）。先前对αCTD的突变分析表明，αCTD中Y263的取代严重损害了Spx激活的转录。用丙氨酸取代αCTD的R261、R268、R289、E255、E298和K294的尝试均未成功，这表明这些残基是必不可少的。为了确定这些RpoA残基对于Spx-RNAP的有效相互作用是否必需，我们在突变体中异位表达了推测具有致死性的突变等位基因，其中“”表示由等位基因突变导致的氨基酸变化。通过互补分析，我们表明与Spx结合的αCTD氨基酸残基对于Spx激活的转录不是必需的，但R261A、E298A和E255A突变体在Spx控制基因的NaCl应激诱导中存在部分缺陷。此外，表达的菌株在抗二硫键应激方面存在缺陷，并且产生的RNAP对Spx的亲和力降低。E255残基对应于αD259，它与αCTD-σ相互作用有关（σR603，对应于σ的R362）。然而，和的联合突变对Spx依赖性表达具有累加的负面影响，这表明这些残基在Spx激活的转录中具有不同的作用。我们的研究结果表明，虽然αCTD对于Spx激活的转录是必需的，但Spx是Spx-αCTD复合物的主要DNA结合决定因素。尽管在中对其进行了广泛研究，但αCTD在激活剂刺激的转录中的作用在中很大程度上尚未得到表征。在这里，我们对推测具有致死性的αCTD丙氨酸密码子取代突变体进行表型分析，以确定这些残基是否在Spx-αCTD-DNA界面的特异性DNA结合中发挥作用。我们的研究结果表明，多亚基RNAP与Spx的接触对于激活是最佳的，而Spx满足上游启动子结合的最严格要求。此外，本研究中靶向诱变的几个αCTD残基在许多细菌物种中是保守的，因此本文可能会对它们在其他调节系统中的功能提供见解。
