Structural and mechanistic basis of σ-dependent transcriptional pausing

Pukhrambam, Chirangini, Molodtsov, Vadim, Kooshkbaghi, Mahdi, Tareen, Ammar, Vu, Hoa, Skalenko, Kyle S, Su, Min, Zhou, Yin, Winkelman, Jared T, Kinney, Justin B, Ebright, Richard H, Nickels, Bryce E (January 2022) Structural and mechanistic basis of σ-dependent transcriptional pausing. BioRxiv. (Unpublished)

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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.

Item Type:	Paper
Subjects:	bioinformatics Investigative techniques and equipment > microscopy > Cryo-electron microscopy bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > transcription bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics Investigative techniques and equipment bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > RNA polymerase Investigative techniques and equipment > microscopy
CSHL Authors:	Kooshkbaghi, Mahdi Tareen, Ammar Kinney, Justin B.
Communities:	CSHL labs > Kinney lab
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	25 January 2022
Date Deposited:	03 Feb 2022 18:17
Last Modified:	16 Jan 2024 21:21
URI:	https://repository.cshl.edu/id/eprint/40510

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