Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts.
Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts.
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DOI：
10.1093/nar/gkm085
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发表时间：
2007
影响因子：
14.9
通讯作者：
Wagner R
中科院分区：
文献类型：
--
作者：
Gildehaus N;Neusser T;Wurm R;Wagner R
大肠杆菌 6S RNA 代表一种非编码 RNA (ncRNA)，基于保守的二级结构和之前的功能研究，有人认为它会干扰转录。描述了 sigma-70 全酶的选择性抑制，优先在延伸的 -10 启动子处，但没有描述稳定期特异性转录，这表明 6S RNA 在从指数期到稳定期的转变中具有直接作用。为了阐明其潜在机制，我们通过凝胶阻滞和交联分析了 6S RNA 与不同形式 RNA 聚合酶的相互作用。证实了 6S RNA 与 Eσ70 的优先结合，但也观察到与 Eσ38 的较弱结合。交联分析揭示了中央 6S RNA 序列元件与 β/β' 和 σ 亚基之间的直接接触。使用指数期和稳定期特异性启动子以及相应的全酶进行的启动子复合物形成和体外转录分析表明，6S RNA 干扰转录起始，但通常不区分指数期和稳定期特异性启动子。此外，我们首次证明 6S RNA 在没有 DNA 的情况下可以作为特定 RNA 分子转录的模板。总之，这项研究揭示了 6S RNA 功能的新方面。
Escherichia coli 6S RNA represents a non-coding RNA (ncRNA), which, based on the conserved secondary structure and previous functional studies, had been suggested to interfere with transcription. Selective inhibition of sigma-70 holoenzymes, preferentially at extended −10 promoters, but not stationary-phase-specific transcription was described, suggesting a direct role of 6S RNA in the transition from exponential to stationary phase. To elucidate the underlying mechanism, we have analysed 6S RNA interactions with different forms of RNA polymerase by gel retardation and crosslinking. Preferred binding of 6S RNA to Eσ70 was confirmed, however weaker binding to Eσ38 was also observed. The crosslinking analysis revealed direct contact between a central 6S RNA sequence element and the β/β′ and σ subunits. Promoter complex formation and in vitro transcription analysis with exponential- and stationary-phase-specific promoters and the corresponding holoenzymes demonstrated that 6S RNA interferes with transcription initiation but does not generally distinguish between exponential- and stationary-phase-specific promoters. Moreover, we show for the first time that 6S RNA acts as a template for the transcription of defined RNA molecules in the absence of DNA. In conclusion, this study reveals new aspects of 6S RNA function.
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