Universität Regensburg, Regensburg Center of Biochemistry (RCB), Lehrstuhl Biochemie III, Regensburg, Germany.
Universität Regensburg, Regensburg Center of Biochemistry (RCB), Lehrstuhl Biochemie III, Regensburg, Germany.
J Biol Chem. 2022 May;298(5):101862. doi: 10.1016/j.jbc.2022.101862. Epub 2022 Mar 25.
Elongating nuclear RNA polymerases (Pols) frequently pause, backtrack, and are then reactivated by endonucleolytic cleavage. Pol backtracking and RNA cleavage are also crucial for proofreading, which contributes to transcription fidelity. RNA polymerase I (Pol I) of the yeast Saccharomyces cerevisiae synthesizes exclusively 35S rRNA, the precursor transcript of mature ribosomal 5.8S, 18S, and 25S rRNA. Pol I contains the specific heterodimeric subunits Rpa34.5/49 and subunit Rpa12.2, which have been implicated in RNA cleavage and elongation activity, respectively. These subunits are associated with the Pol I lobe structure and encompass different structural domains, but the contribution of these domains to RNA elongation is unclear. Here, we used Pol I mutants or reconstituted Pol I enzymes to study the effects of these subunits and/or their distinct domains on RNA cleavage, backtracking, and transcription fidelity in defined in vitro systems. Our findings suggest that the presence of the intact C-terminal domain of Rpa12.2 is sufficient to support the cleavage reaction, but that the N-terminal domains of Rpa12.2 and the heterodimer facilitate backtracking and RNA cleavage. Since both N-terminal and C-terminal domains of Rpa12.2 were also required to faithfully incorporate NTPs in the growing RNA chain, efficient backtracking and RNA cleavage might be a prerequisite for transcription fidelity. We propose that RNA Pols containing efficient RNA cleavage activity are able to add and remove nucleotides until the matching nucleotide supports RNA chain elongation, whereas cleavage-deficient enzymes can escape this proofreading process by incorporating incorrect nucleotides.
延伸核 RNA 聚合酶（Pols）经常暂停、回溯，然后通过核酸内切酶切割重新激活。Pol 回溯和 RNA 切割对于校对也至关重要，校对有助于转录保真度。酵母酿酒酵母的 RNA 聚合酶 I（Pol I）仅合成 35S rRNA，这是成熟核糖体 5.8S、18S 和 25S rRNA 的前体转录物。Pol I 包含特定的异二聚体亚基 Rpa34.5/49 和亚基 Rpa12.2，它们分别参与 RNA 切割和延伸活性。这些亚基与 Pol I 叶状结构相关，并包含不同的结构域，但这些结构域对 RNA 延伸的贡献尚不清楚。在这里，我们使用 Pol I 突变体或重组 Pol I 酶在定义明确的体外系统中研究这些亚基及其不同结构域对 RNA 切割、回溯和转录保真度的影响。我们的研究结果表明，完整的 Rpa12.2 C 末端结构域的存在足以支持切割反应，但 Rpa12.2 的 N 末端结构域和异二聚体有利于回溯和 RNA 切割。由于 Rpa12.2 的 N 末端和 C 末端结构域对于在生长的 RNA 链中准确掺入 NTP 也是必需的，因此有效的回溯和 RNA 切割可能是转录保真度的前提。我们提出，含有有效 RNA 切割活性的 RNA Pol 能够添加和去除核苷酸，直到匹配的核苷酸支持 RNA 链延伸，而缺乏切割活性的酶可以通过掺入错误的核苷酸来逃避这个校对过程。
