During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded 'transcription bubble,' and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA 'scrunching' and 'anti-scrunching,' the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.

在转录起始过程中，RNA聚合酶（RNAP）与启动子DNA结合，解开启动子DNA形成一个含有单链“转录气泡”的RNAP-启动子开放复合物（RPo），并选择一个转录起始位点（TSS）。根据启动子序列和起始底物浓度，TSS选择发生在启动子区域内的不同位置。已提出TSS选择的可变性涉及DNA的``卷曲''和``反卷曲''，其标志是：（i）RNA相对于DNA的前缘而不是后缘的向前和向后运动，和（ ii）转录气泡的膨胀和收缩。在这里，使用体外和体内蛋白质-DNA光交联和单分子纳米操作，我们显示细菌TSS选择既展现出抗扭力又具有抗扭力的标志，并且我们定义了抗扭力和抗扭力的能量。结果建立了细菌RNAP选择TSS的机制，并提出了细菌，古细菌和真核RNAP选择TSS的一般机制。