Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854.
Department of Genetics, Rutgers University, Piscataway, NJ 08854.
Proc Natl Acad Sci U S A. 2022 Jun 7;119(23):e2201301119. doi: 10.1073/pnas.2201301119. Epub 2022 Jun 2.
In σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein–DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway “backtracked” states that are substrates for the transcript-cleavage factors of the Gre family and on-pathway “scrunched” states that mediate pause escape. Here, using site-specific protein–DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR′ promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2–4 bp, and σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2–3 bp. Analogous experiments with a library of 47 (∼16,000) transcribed-region sequences show that the state scrunched by 2–3 bp—and only that state—is associated with the consensus sequence, T−3N−2Y−1G+1, (where −1 corresponds to the position of the RNA 3′ end), which is identical to the consensus for pausing in initial transcription and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T−3 resides in the DNA nontemplate strand. A cryoelectron microscopy structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T−3 of the consensus sequence exerts its effects by facilitating scrunching.
在 σ 依赖的转录暂停中，转录起始因子 σ 与 RNA 聚合酶（RNAP）一起转位，与转录区中的类似启动子的序列元件进行序列特异性的蛋白-DNA 相互作用，诱导暂停。有人提出，在 σ 依赖的暂停中，RNAP 活性中心可以进入非途径“回溯”状态，这些状态是 Gre 家族的转录物切割因子的底物，并且可以进入途径“压缩”状态，从而介导暂停逃逸。在这里，我们使用特异性蛋白-DNA 光交联来定义 RNAP 尾部和头部以及 σ 相对于 λPR′启动子 DNA 的位置，直接表明体外无 GreB 存在时 σ 依赖的暂停主要涉及回溯 2-4bp 的状态，并且体外和体内有 GreB 存在时 σ 依赖的暂停主要涉及回溯 2-3bp 的状态。类似的实验中，我们使用了一个由 47 个（约 16000 个）转录区序列组成的文库，表明 2-3bp 压缩的状态——而且只有这种状态——与共识序列 T−3N−2Y−1G+1 相关联，其中-1 对应于 RNA 3' 端的位置），这与初始转录暂停的共识序列相同，并且与转录延伸暂停的共识序列相关。用异源双链模板进行的实验表明，位置 T−3 的序列信息位于 DNA 非模板链上。一个参与 σ 依赖暂停的复合物的低温电子显微镜结构揭示了 DNA 非模板和模板链上 DNA 压缩的位置，并表明共识序列中位置 T−3 通过促进压缩来发挥其作用。
