Sigma38 (rpoS) RNA polymerase promoter engagement via-10 region on nucleotides
Sigma38 (rpoS) RNA polymerase promoter engagement via-10 region on nucleotides
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DOI：
10.1074/jbc.m102886200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Gralla, JD
中科院分区：
文献类型：
--
作者：
Lee, SJ;Gralla, JD
使用模拟封闭和开放复合物的DNA探针的带移分析被用来探索由sigma 38（rpoS）RNA聚合酶识别启动子的决定因素。发现双链体识别比在sigma 70启动子使用中观察到的要弱得多。然而，与试图模拟熔化的DNA的叉连接探针的结合非常强。这种结合通过非模板链发生，其中两个保守的连接核苷酸（-12T和-11A）的身份是最重要的。经修饰的启动子共有序列将这两个核苷酸鉴定为仅四个高度保守的核苷酸（加下划线的）之一，并且所有四个都在-10区域（CTAcacT从-13到-7）中。剩余的两个核苷酸显示出具有不同的作用，-13C在防止异源σ 70聚合酶的识别中，-7T在指导酶异构化中。这些-10区域核苷酸似乎在秋季解链之前具有其主要功能，因为具有解链起始位点的探针对这些位置处的取代相对不敏感。这些结果表明sigma 38机制不同于sigma 70机制，并且这种差异可能有助于在静止期存在的条件下选择性使用sigma 38。
Band shift assays using DNA probes that mimic closed and open complexes were used to explore the determinants of promoter recognition by sigma38 (rpoS) RNA polymerase. Duplex recognition was found to be much weaker than that observed in sigma70 promoter usage. However, binding to fork junction probes, which attempt to mimic melted DNA, was very strong. This binding occurs via the non-template strand with the identity of the two conserved junction nucleotides (-12T and -11A) being of paramount importance. A modified promoter consensus sequence identified these two nucleotides as among only four (underlined) that are highly conserved, and all four were in the -10 region (CTAcacT from -13 to -7). The remaining two nucleotides were shown to have different roles, -13C in pre-venting recognition by the heterologous sigma70 polymerase and -7T in directing enzyme isomerization. These -10 region nucleotides appear to have their primary function prior to fall melting because probes that had a melted start site were relatively insensitive to substitution at these positions. These results suggest the sigma38 mechanism differs from the sigma70 mechanism, and this difference likely contributes to selective use of sigma38 under conditions that exist during stationery phase.