Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans
Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans
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DOI：
10.1128/jb.00155-11
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发表时间：
2011-07-01
影响因子：
3.2
通讯作者：
Goodman, Steven D.
中科院分区：
文献类型：
--
作者：
Hung, David C. I.;Downey, Jennifer S.;Goodman, Steven D.
在变形链球菌中，能力和细菌素的产生均由 ComC 和 ComED 双组分信号转导系统控制。最近对变形链球菌的研究表明，纯化的 ComE 与 nlmC-comC 启动子区域中的两个 11 bp 同向重复序列结合，其中 ComE 激活 nlmC 并抑制 comC。在这项工作中，进行了定量结合研究和 DNase I 足迹分析，以计算平衡解离常数并进一步表征 ComE 的结合位点。我们发现 ComE 保护包含两个同向重复序列的序列，具有纳摩尔范围内的平衡解离常数，并协同结合这两个同向重复序列。此外，在 cslAB、comED、comX、ftf、vicRKX、gtfD、gtfB、gtfC 和 gbpB 的上游发现了类似的同向重复序列。对这些序列中的每一个进行了定量结合研究，结果表明，只有 cslAB 对 ComE 具有与 comC 上游区域相似的特异性和高亲和力。结合序列的突变分析表明，ComE 不需要两个重复序列就能以高亲和力结合 DNA，这表明基因组中的单位点序列可能是 ComE 介导的调节的目标。基于突变分析和 DNase I 足迹分析，我们提出了一个一致的 ComE 结合位点 TCBTAAAYSGT。
In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.