E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase.
E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase.
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DOI：
10.1016/j.jmb.2010.03.051
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发表时间：
2010-05-28
影响因子：
5.6
通讯作者：
Block SM
中科院分区：
文献类型：
--
作者：
Herbert KM;Zhou J;Mooney RA;Porta AL;Landick R;Block SM
NusG是E.大肠杆菌，能够增加总的转录速率。RNA聚合酶（RNAP）的转录延长经常被不同持续时间的暂停中断，已知NusG至少会减少一些暂停状态的占用。然而，NusG是否主要通过（1）增加停顿之间的延长速率、（2）减少停顿的寿命、或（3）减少进入停顿状态的速率来增强转录尚未确定。在这里，我们研究了转录的单分子RNAP在各种条件下的NTP浓度，施加负载，和温度，使用光捕获检测能够区分暂停短暂的1秒。我们发现NusG增加了伸长率，即，沿模板沿着的无停顿速度。由于暂停是与延长竞争的非通路状态，我们观察到进入短寿命暂停状态的速率随之降低。然而，对短暂停顿和延长的影响相对温和。更引人注目的是NusG抑制进入长寿命（“稳定”）暂停的效果。由于典型基因转录所需的时间中有很大一部分可能被长时间的停顿所占据，NusG能够对RNA合成速率产生显著的调节作用。观察到的NusG特性与统一模型一致，其中该辅助因子的功能是促进酶沿着DNA模板的转录下游运动，其具有将RNAP从易位前状态正向偏向易位后状态的作用。
NusG is an essential transcription factor in E. coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states. Here, we studied transcription by single molecules of RNAP under various conditions of NTP concentration, applied load, and temperature, using an optical trapping assay capable of distinguishing pauses as brief as 1 s. We found that NusG increases the rate of elongation, i.e., the pause-free velocity along the template. Because pauses are off-pathway states that compete with elongation, we observed a concomitant decrease in the rate of entry into short-lifetime, paused states. The effects on short pauses and elongation were comparatively modest, however. More dramatic was the effect of NusG on suppressing entry into long-lifetime (“stabilized”) pauses. Because a significant fraction of the time required for the transcription of a typical gene may be occupied by long pauses, NusG is capable of exerting a significant modulatory effect on the rates of RNA synthesis. The observed properties of NusG were consistent with a unified model where the function of this accessory factor is to promote transcriptionally downstream motion of the enzyme along the DNA template, which has the effect of forward-biasing RNAP from the pre-translocated state towards the post-translocated state.
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