Binding and release of the 6S transcriptional control RNA
Binding and release of the 6S transcriptional control RNA
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DOI：
10.1261/rna.2036210
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发表时间：
2010-05-01
期刊：
影响因子：
4.5
通讯作者：
Unrau, Peter J.
中科院分区：
文献类型：
--
作者：
Shephard, Lindsay;Dobson, Neil;Unrau, Peter J.
6S RNA是一种重要的非编码RNA，调节真细菌的转录。在大肠杆菌中，这种RNA与sigma（70）RNA聚合酶全酶结合，并通过合成短产物RNA而释放。为了确定6S RNA序列如何控制结合和释放，我们使用体外选择来筛选含有功能性6S RNA变体的类似于4 × 10（12）序列的高多样性文库。发现结合的关键残基位于6S RNA转录泡模拟结构上游的“-35”区域。突变这些在遗传学上保守的残基总是导致结合的减少，并且去除它们消除了结合，暗示这些核苷酸与E sigma（70）复合物在生物学上重要的相互作用。有趣的是，发现也在pRNA合成位点上游的遗传学保守的“-10”残基的突变除了调节-35结合之外还影响6S RNA释放速率。这些结果表明，6S RNA-35与sigma（70）RNA聚合酶全酶的结合如何调节“强”和“弱”-35 DNA启动子的表达，并表明6S RNA释放速率已随进化时间进行微调，以便正确调节细胞转录水平。
6S RNA is an important noncoding RNA that regulates eubacterial transcription. In Escherichia coli this RNA binds to the sigma(70) RNA polymerase holoenzyme and is released by the synthesis of a short product RNA. In order to determine how binding and release are controlled by the 6S RNA sequence, we used in vitro selection to screen a high diversity library containing similar to 4 x 10(12) sequences for functional 6S RNA variants. Residues critical for binding were found to be located in a "-35'' region upstream of the 6S RNA transcription bubble mimic structure. Mutating these phylogenetically conserved residues invariably led to decreases in binding and removing them abolished binding, implicating these nucleotides in a biologically important interaction with the E sigma(70) complex. Interestingly, mutation of phylogenetically conserved "-10'' residues that were also upstream of the site of pRNA synthesis was found to influence 6S RNA release rates in addition to modulating -35 binding. These results indicate how 6S RNA -35 binding to sigma(70) RNA polymerase holoenzyme can regulate expression from "strong'' and "weak'' -35 DNA promoters and suggest that 6S RNA release rates have been fine tuned over evolutionary time so as to correctly regulate cellular levels of transcription.