Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3′ end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3′ end of RNA within the active center. We show that the TL is required to transfer the 3′ end of misincorporated transcript from cleavage-inefficient ‘misincorporation site’ into the cleavage-efficient ‘backtracked site’, where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.

中文翻译：

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RNA 聚合酶回溯的两种不同途径决定了 RNA 水解过程中对触发环的要求

转录 RNA 聚合酶 (RNAP) 会陷入回溯，即转录本 3' 端与模板 DNA 脱离时的现象。回溯是由核酸序列或错误核苷酸的错误掺入引起的。要恢复生产性延伸，必须通过 RNA 水解来解决回溯复合物。目前对于大肠杆菌 RNAP 催化该反应的机制尚未达成共识。在这里，我们使用盐酰胺 A，我们发现它抑制 RNAP 催化结构域触发环 (TL)，以表明在错误掺入事件的校对期间 RNA 切割需要 TL，但在序列依赖性回溯复合物的切割过程中几乎没有作用。结果表明，由错误掺入引起的回溯不同于序列相关的回溯，导致活性中心内 RNA 3'端的不同构象。我们表明，需要 TL 将错误掺入的转录物的 3' 端从切割效率低下的“错误掺入位点”转移到切割效率高的“回溯位点”，水解通过转录辅助催化发生，并且在很大程度上独立于 TL . 这些发现解决了围绕大肠杆菌 RNA 聚合酶水解 RNA 机制的争议，并表明 TL 在 RNA 切割中的作用在细菌之间存在差异。其中水解通过转录辅助催化发生，并且在很大程度上独立于 TL。这些发现解决了围绕大肠杆菌 RNA 聚合酶水解 RNA 机制的争议，并表明 TL 在 RNA 切割中的作用在细菌之间存在差异。其中水解通过转录辅助催化发生，并且在很大程度上独立于 TL。这些发现解决了围绕大肠杆菌 RNA 聚合酶水解 RNA 机制的争议，并表明 TL 在 RNA 切割中的作用在细菌之间存在差异。