Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russia.
Department of Natural Sciences, Novosibirsk State University, Novosibirsk 630090, Russia.
Int J Mol Sci. 2024 Nov 14;25(22):12190. doi: 10.3390/ijms252212190.
The mechanism of transcription proceeds through the formation of R-loop structures containing a DNA-RNA heteroduplex and a single-stranded DNA segment that should be placed inside the elongation complex; therefore, these nucleic acid segments are limited in length. The attachment of each nucleotide to the 3' end of an RNA strand requires a repeating cycle of incoming nucleoside triphosphate binding, catalysis, and enzyme translocation. Within these steps of transcription elongation, RNA polymerase sequentially goes through several states and is post-translocated, catalytic, and pre-translocated. Moreover, the backward movement of the polymerase, which is essential for transcription pausing and proofreading activity, gives rise to a backtracked state. In the present study, to analyze both the efficacy of transcription elongation complex (TEC) formation and the rate of RNA synthesis, we used a set of model R-loops that mimic the pre-translocated state, post-translocated state, backtracked state, and a misincorporation event. It was shown that TEC assembly proceeds as an equilibrium process, including the simultaneous formation of a catalytically competent TEC as well as a catalytically inactive conformation. Our data suggest that the inactive complex of RNA polymerase with an R-loop undergoes slow conformational changes, resulting in a catalytically competent TEC. It was revealed that the structural features of R-loops affect the ratio between active and inactive states of the TEC, the rate of conformational rearrangements required for the induced-fit transition from the inactive state to the catalytically competent TEC, and the rates of accumulation of both the total RNA products and long RNA products.
转录的机制是通过形成含有 DNA-RNA 杂合双链和单链 DNA 片段的 R 环结构来进行的，该单链 DNA 片段应置于延伸复合物内部；因此，这些核酸片段的长度受到限制。每个核苷酸与 RNA 链 3' 端的连接需要重复循环结合、催化和酶转位。在转录延伸的这些步骤中，RNA 聚合酶依次经历几个状态，并经历后转位、催化和预转位。此外，聚合酶的向后运动对于转录暂停和校对活性至关重要，导致了回溯状态。在本研究中，为了分析转录延伸复合物 (TEC) 的形成效率和 RNA 合成速率，我们使用了一组模型 R 环，模拟了预转位状态、后转位状态、回溯状态和一个错配事件。结果表明，TEC 的组装是一个平衡过程，包括同时形成具有催化能力的 TEC 以及无催化活性的构象。我们的数据表明，与 R 环结合的无催化活性的 RNA 聚合酶复合物经历缓慢的构象变化，导致具有催化能力的 TEC。结果表明，R 环的结构特征影响 TEC 的活性和无活性状态之间的比例、诱导契合从无活性状态到具有催化能力的 TEC 的构象重排所需的速率，以及总 RNA 产物和长 RNA 产物的积累速率。
