7N4E

Escherichia coli sigma 70-dependent paused transcription elongation complex

Summary for 7N4E

• Entry DOI: 10.2210/pdb7n4e/pdb
• EMDB information: 24148
• Descriptor: DNA (61-MER), MAGNESIUM ION, DNA-directed RNA polymerase subunit alpha, ... (10 entities in total)
• Functional Keywords: elongation, pausing, sigma 70, transcription complex, dna scrunching, transferase-dna-rna complex, transferase/dna/rna
• Biological source: Escherichia coli; More
• Total number of polymer chains: 9
• Total formula weight: 501403.34

Authors

Molodtsov, V.,Su, M.,Ebright, R.H. (deposition date: 2021-06-04, release date: 2022-06-15, Last modification date: 2024-05-29)

Primary citation

Pukhrambam, C.,Molodtsov, V.,Kooshkbaghi, M.,Tareen, A.,Vu, H.,Skalenko, K.S.,Su, M.,Yin, Z.,Winkelman, J.T.,Kinney, J.B.,Ebright, R.H.,Nickels, B.E.
Structural and mechanistic basis of sigma-dependent transcriptional pausing.
Proc.Natl.Acad.Sci.USA, 119:e2201301119-e2201301119, 2022

PubMed 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 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.

PubMed: 35653571
DOI: 10.1073/pnas.2201301119

Experimental method

ELECTRON MICROSCOPY (3.8 Å)