RNA polymerase SI3 domain modulates global transcriptional pausing and pause-site fluctuations.

Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE

Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.

DNA-Directed RNA Polymerases*/chemistry ; DNA-Directed RNA Polymerases*/genetics ; DNA-Directed RNA Polymerases*/metabolism ; Escherichia coli*/genetics ; Escherichia coli*/metabolism ; Escherichia coli Proteins*/metabolism ; Escherichia coli Proteins*/genetics ; Transcription, Genetic*; Gene Expression Regulation, Bacterial ; Protein Domains

Transcriptional pausing aids gene regulation by cellular RNA polymerases (RNAPs). A surface-exposed domain inserted into the catalytic trigger loop (TL) of Escherichia coli RNAP, called SI3, modulates pausing and is essential for growth. Here we describe a viable E. coli strain lacking SI3 enabled by a suppressor TL substitution (β'Ala941→Thr; ΔSI3*). ΔSI3* increased transcription rate in vitro relative to ΔSI3, possibly explaining its viability, but retained both positive and negative effects of ΔSI3 on pausing. ΔSI3* inhibited pauses stabilized by nascent RNA structures (pause hairpins; PHs) but enhanced other pauses. Using NET-seq, we found that ΔSI3*-enhanced pauses resemble the consensus elemental pause sequence whereas sequences at ΔSI3*-suppressed pauses, which exhibited greater association with PHs, were more divergent. ΔSI3*-suppressed pauses also were associated with apparent pausing one nucleotide upstream from the consensus sequence, often generating tandem pause sites. These '-2 pauses' were stimulated by pyrophosphate in vitro and by addition of apyrase to degrade residual NTPs during NET-seq sample processing. We propose that some pauses are readily reversible by pyrophosphorolysis or single-nucleotide cleavage. Our results document multiple ways that SI3 modulates pausing in vivo and may explain discrepancies in consensus pause sequences in some NET-seq studies.
(© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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K99 AI166036 United States AI NIAID NIH HHS; R01 GM038660 United States GM NIGMS NIH HHS; R37 GM038660 United States GM NIGMS NIH HHS; GM38660 United States NH NIH HHS

EC 2.7.7.6 (DNA-Directed RNA Polymerases)
0 (Escherichia coli Proteins)
EC 2.7.7.- (beta' subunit of RNA polymerase)

Date Created: 20240330 Date Completed: 20240508 Latest Revision: 20240803

20240803

PMC11077087

10.1093/nar/gkae209

38554114