Department of Biochemistry, Brandeis University, Waltham, United States.
Department of Biochemistry, University of Wisconsin, Madison, United States.
Elife. 2019 Feb 5;8:e40576. doi: 10.7554/eLife.40576.
RNA polymerases (RNAPs) contain a conserved 'secondary channel' which binds regulatory factors that modulate transcription initiation. In , the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a 'delayed inhibition' mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.
RNA 聚合酶（RNAPs）含有一个保守的“次要通道”，该通道结合调节转录起始的调节因子。在，二级通道因子（SCFs）GreB 和 DksA 都抑制核糖体 RNA（rRNA）转录，但 SCF 加载和抑制机制尚不清楚。我们在体外观察到荧光标记的 GreB 分子与单个 RNAP 结合，并从 rRNA 启动子起始单个转录物。GreB 仅在与 RNAP 形成复合物的情况下才能到达和离开启动子。GreB 没有改变初始 RNAP-启动子的结合，但相反阻止了初始 RNAP-启动子复合物构象重排后的一个步骤。引人注目的是，GreB-RNAP 复合物从未在 rRNA 启动子处起始；只有到达启动子而没有结合 GreB 的 RNAP 分子才能产生转录物。数据表明，一种模型 SCF 通过“延迟抑制”机制发挥作用，并表明 rRNA 启动子被 GreB/DksA 抑制，因为它们寿命短暂的 RNAP 复合物不允许足够的时间让 SCF 解离。
