Key features of σS required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme
Key features of σS required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme
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DOI：
10.1073/pnas.1311642110
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发表时间：
2013-10-01
影响因子：
11.1
通讯作者：
Gourse, Richard L.
中科院分区：
文献类型：
--
作者：
Banta, Amy B.;Chumanov, Robert S.;Gourse, Richard L.
细菌使用多个西格玛因子来协调基因表达，以应对环境扰动。在大肠杆菌和其他蛋白质细菌中，转录因子CR1通过促进Sigma(5)与核心核糖核酸聚合酶的结合，在细胞应激期间刺激依赖于Sigma(S)的转录。CRL特异性识别sigma(5)，而不是同源的、更丰富的初级sigma因子sigma(70)的分子基础尚不清楚。在这里，我们使用体内细菌双杂交分析和体外对苯甲酰苯丙氨酸交联来确定S、S负责CRL特异性识别的特征。我们在S S保守结构域2(sigma(S)(2))中发现了充分必要条件，使CRL能够识别sigma(70)保守结构域2，一个在启动子熔融区附近，另一个在一个大的非保守区中断sigma(70)序列的位置。然后，我们使用发光共振能量转移来直接证明CRL利用西格玛(5)上的这些特异性决定因素促进全酶组装。我们的结果解释了CRL如何区分大部分同源的sigma因子，并激活离散的启动子集，即使它不与启动子DNA结合。
Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other.-proteobacteria, the transcription factor Crl stimulates sigma(s) -dependent transcription during times of cellular stress by promoting the association of sigma(5) with core RNA polymerase. The molecular basis for specific recognition of sigma(5) by Crl, rather than the homologous and more abundant primary sigma factor sigma(70), is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoylphenylalanine cross-linking in vitro to define the features in s S responsible for specific recognition by Crl. We identify residues in s S conserved domain 2 (sigma(S) (2)) that are necessary and sufficient to allow recognition of sigma(70) conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of sigma(70.) We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on sigma(5). Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.