Closing and opening of the RNA polymerase trigger loop
Closing and opening of the RNA polymerase trigger loop
复制标题
DOI：
10.1073/pnas.1920427117
复制
发表时间：
2020-07-07
影响因子：
11.1
通讯作者：
Ebright, Richard H.
中科院分区：
文献类型：
--
作者：
Mazumder, Abhishek;Lin, Miaoxin;Ebright, Richard H.
RNA聚合酶（RNAP）触发环（TL）是RNAP活性中心的移动结构元件，基于晶体结构，有人提出在允许NTP底物进入活性中心的“未折叠”/“开放”状态和将NTP底物保持在活性中心的“折叠”/“闭合”状态之间循环。在这里，通过量化 TL 中的第一个荧光探针与 RNAP 或 DNA 中其他位置的第二个荧光探针之间的单分子荧光共振能量转移，我们检测并表征了溶液中 TL 的关闭和打开。我们展示了 TL 在毫秒时间尺度上关闭和打开；我们表明，TL 关闭和打开为 NTP 互补性、NTP 核糖/脱氧核糖同一性和 NTP 三/二/单磷酸同一性提供了检查点，并可作为抑制剂的靶点；我们发现，TL 关闭和打开的一个循环通常发生在转录延伸的每个核苷酸添加循环中。
The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.