University of Turku, Department of Biochemistry, FIN-20014 Turku, Finland.
Nucleic Acids Res. 2018 Nov 16;46(20):10870-10887. doi: 10.1093/nar/gky883.
All cellular RNA polymerases (RNAP) occasionally backtrack along the template DNA as part of transcriptional proofreading and regulation. Here, we studied the mechanism of RNAP backtracking by one nucleotide using two complementary approaches that allowed us to precisely measure the occupancy and lifetime of the backtracked state. Our data show that the stability of the backtracked state is critically dependent on the closure of the RNAP active site by a mobile domain, the trigger loop (TL). The lifetime and occupancy of the backtracked state measurably decreased by substitutions of the TL residues that interact with the nucleoside triphosphate (NTP) substrate, whereas amino acid substitutions that stabilized the closed active site increased the lifetime and occupancy. These results suggest that the same conformer of the TL closes the active site during catalysis of nucleotide incorporation into the nascent RNA and backtracking by one nucleotide. In support of this hypothesis, we construct a model of the 1-nt backtracked complex with the closed active site and the backtracked nucleotide in the entry pore area known as the E-site. We further propose that 1-nt backtracking mimics the reversal of the NTP substrate loading into the RNAP active site during on-pathway elongation.
所有细胞 RNA 聚合酶（RNAP）偶尔会沿着模板 DNA 回溯，这是转录校对和调控的一部分。在这里，我们使用两种互补的方法研究了 RNAP 一个核苷酸回溯的机制，这使我们能够精确测量回溯状态的占有率和寿命。我们的数据表明，回溯状态的稳定性严重依赖于可移动结构域，即触发环（TL），其封闭 RNAP 活性位点。与核苷三磷酸（NTP）底物相互作用的 TL 残基的取代可显著降低回溯状态的寿命和占有率，而稳定封闭活性位点的氨基酸取代则增加了寿命和占有率。这些结果表明，TL 的相同构象在将核苷酸掺入新生 RNA 的催化过程中和通过一个核苷酸回溯时关闭活性位点。为了支持这一假设，我们构建了一个具有封闭活性位点和位于入口孔区域（称为 E 位）的 1 个核苷酸回溯核苷酸的 1-nt 回溯复合物模型。我们进一步提出，1-nt 回溯模拟了 NTP 底物在途径延伸过程中加载到 RNAP 活性位点的逆转。
