Phosphorylation of the group A streptococcal CovR response regulator causes dimerization and promoter-specific recruitment by RNA polymerase
Phosphorylation of the group A streptococcal CovR response regulator causes dimerization and promoter-specific recruitment by RNA polymerase
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DOI：
10.1128/jb.00198-06
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发表时间：
2006-07-01
影响因子：
3.2
通讯作者：
Scott, June R.
中科院分区：
文献类型：
--
作者：
Gusa, Asiya A.;Gao, Jinxin;Scott, June R.
A组链球菌（GAS），化脓性链球菌，是一种重要的人类病原体，其引起从自限性咽炎到与显著发病率和死亡率相关的严重侵入性疾病的严重程度不等的感染。GAS的致病作用是由毒力因子的表达介导的，其中之一是透明质酸胶囊（由has操纵子中的基因编码）。这些毒力因子的表达由GAS的CovR/S（CsrR/S）双组分调节系统控制，该系统直接或间接地调节约15%的基因组的表达。CovR是OmpR/PhoB转录调节因子家族的成员。在这里，我们表明，磷酸乙酰导致CovR的二聚化。使用CovR的D53 A突变体未观察到二聚化，表明D53是CovR中的磷酸化位点。磷酸化刺激CovR与含有has操纵子（Phas）启动子的DNA片段的结合大约两倍。CovR D53 A突变蛋白与Phas的结合与野生型未磷酸化CovR的结合不可区分。在体外转录，使用纯化的GAS RNA聚合酶，表明野生型CovR抑制转录，抑制刺激超过六倍的磷酸化。在RNA聚合酶的存在下，在磷酸化的Phas结合，但不是未磷酸化的，CovR被刺激约四倍，这解释了磷酸化对阻遏与DNA结合的影响的差异。因此，CovR对Phas的调节是直接的，并且Phas的阻遏程度由CovR的磷酸化控制。
The group A streptococcus (GAS), Streptococcus pyogenes, is an important human pathogen that causes infections ranging in severity from self-limiting pharyngitis to severe invasive diseases that are associated with significant morbidity and mortality. The pathogenic effects of GAS are mediated by the expression of virulence factors, one of which is the hyaluronic acid capsule (encoded by genes in the has operon). The expression of these virulence factors is controlled by the CovR/S (CsrR/S) two-component regulatory system of GAS which regulates, directly or indirectly, the expression of about 15% of the genome. CovR is a member of the OmpR/PhoB family of transcriptional regulators. Here we show that phosphorylation by acetyl phosphate results in dimerization of CovR. Dimerization was not observed using a D53A mutant of CovR, indicating that D53 is the site of phosphorylation in CovR. Phosphorylation stimulated binding of CovR to a DNA fragment containing the promoter of the has operon (Phas) approximately twofold. Binding of CovR D53A mutant protein to Phas was indistinguishable from the binding of wild-type unphosphorylated CovR. In vitro transcription, using purified GAS RNA polymerase, showed that wild-type CovR repressed transcription, and repression was stimulated more than sixfold by phosphorylation. In the presence of RNA polymerase, binding at Phas of phosphorylated, but not unphosphorylated, CovR was stimulated about fourfold, which accounts for the difference in the effect of phosphorylation on repression versus DNA binding. Thus, regulation of Phas by CovR is direct, and the degree of repression of Phas is controlled by the phosphorylation of CovR.