Structural and mechanistic basis of σ-dependent transcriptional pausing
Structural and mechanistic basis of σ-dependent transcriptional pausing
Abstract 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 that 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 cryo-EM 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.
Ebright Richard H.、Molodtsov Vadim、Zhou Yin、Winkelman Jared T.、Pukhrambam Chirangini、Skalenko Kyle S.、Kinney Justin B.、Nickels Bryce E.、Tareen Ammar、Kooshbaghi Mahdi、Su Min、Vu Hoa
Waksman Institute of Microbiology, Rutgers University||Department of Chemistry and Chemical Biology, Rutgers UniversityWaksman Institute of Microbiology, Rutgers University||Department of Chemistry and Chemical Biology, Rutgers UniversityWaksman Institute of Microbiology, Rutgers University||Department of Chemistry and Chemical Biology, Rutgers UniversityWaksman Institute of Microbiology, Rutgers University||Department of Genetics, Rutgers University||Department of Chemistry and Chemical Biology, Rutgers UniversityWaksman Institute of Microbiology, Rutgers University||Department of Genetics, Rutgers UniversityWaksman Institute of Microbiology, Rutgers University||Department of Genetics, Rutgers UniversitySimons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring HarborWaksman Institute of Microbiology, Rutgers University||Department of Genetics, Rutgers UniversitySimons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring HarborSimons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring HarborLife Sciences Institute, University of MichiganWaksman Institute of Microbiology, Rutgers University||Department of Genetics, Rutgers University
分子生物学生物物理学生物科学研究方法、生物科学研究技术
RNA polymerasesigmatranscription elongationpausingDNA scrunchingphotocrosslinkingdeep sequencingcryo-EM
Ebright Richard H.,Molodtsov Vadim,Zhou Yin,Winkelman Jared T.,Pukhrambam Chirangini,Skalenko Kyle S.,Kinney Justin B.,Nickels Bryce E.,Tareen Ammar,Kooshbaghi Mahdi,Su Min,Vu Hoa.Structural and mechanistic basis of σ-dependent transcriptional pausing[EB/OL].(2025-03-28)[2025-08-02].https://www.biorxiv.org/content/10.1101/2022.01.24.477500.点此复制
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