Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak-trigger-freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.

中文翻译：

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细菌RNA聚合酶从pyrG和pyrBI启动子重复转录的结构基础。

重复转录是通过RNA聚合酶进行RNA合成的非规范形式，其中DNA模板中单个碱基指定的核糖核苷酸重复添加到新生的RNA转录物中。我们先前确定了从pyrG启动子进行重复转录的细菌RNA聚合酶的X射线晶体结构，该酶包含8个以DNA中的3个C碱基为模板合成的poly-G RNA碱基，并在不置换启动子的情况下扩展了RNA从核心酶中识别出σ因子。在这项研究中，我们使用浸泡触发冻结X射线晶体学确定了pyrG启动子的一系列转录起始复合物结构。我们还进行了生化分析，以监测来自pyrG启动子的RNA合成过程中模板DNA的易位，并进行体外转录分析以确定来自pyrG启动子变体的聚G RNA的长度。我们的研究揭示了RNA如何在模板DNA上滑动以及RNA聚合酶和模板DNA如何确定重复性RNA产物的长度。最后，我们确定了pyrBI启动子上转录起始复合物的结构，并提出了RNA滑移和延伸的另一种机制，该机制要求σ与核心酶解离。