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Nascent elongating transcript sequencing (NET-seq) show different pause registers with apyrase treatment during library preparation

ABSTRACT: Transcriptional pausing aids gene regulation by cellular RNA polymerases (RNAPs). In many bacteria, a surface-exposed domain inserted into the catalytic trigger loop (TL) of RNAP, called SI3 in Escherichia coli, 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; ∆SI3*). ∆SI3* increased transcription rate in vitro relative to ∆SI3, possibly explaining its viability, but retained both positive and negative effects of ∆SI3 on pausing. ∆SI3* inhibited pauses stabilized by nascent RNA structures (pause hairpins; PHs) but enhanced other pauses. Using NET-seq, we found that ∆SI3*-enhanced pauses resemble the consensus elemental pause sequence whereas sequences at ∆SI3*-suppressed pauses, which exhibited greater association with PHs, were more divergent. ∆SI3*-suppressed pauses also were associated with apparent pausing one nt 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.

ORGANISM(S): Escherichia coli Bacillus subtilis

PROVIDER: GSE240163 | GEO | 2023/11/01

REPOSITORIES: GEO

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Nascent elongating transcript sequencing (NET-seq) with SI3 domain-deleted Escherichia coli reveals different pause profiles from wildtype

Project description:Transcriptional pausing is a fundamental mechanism that aids gene regulation by cellular RNA polymerases (RNAPs). In many bacterial lineages, a large, surface-exposed domain inserted into the catalytic trigger loop (TL) of RNAP called sequence insertion 3 (SI3) modulates transcription pausing. However, the in vivo roles of SI3 remain largely unknown due in part to the lethality of SI3 deletion. Here we describe construction of a viable Escherichia coli strain with a complete SI3 deletion enabled by a suppressor missense mutation in the portion of rpoC encoding the TL (ß′A941T; ∆SI3*). The ∆SI3* RNAP exhibited increased transcript elongation rate relative to ∆SI3 RNAP lacking the TL substitution, which may explain viability of the ∆SI3* strain. Using NET-seq, we found that transcriptional pausing in the ∆SI3* strain was increased at some sites and decreased at others compared to wild-type E. coli, except in ribosomal RNA genes. The ∆SI3*-enhanced pauses had a sequence motif similar to the consensus elemental pause sequence whereas ∆SI3*-suppressed pauses had less similarity to the consensus motif and appeared associated with upstream RNA structures thought to stabilize pausing (pause hairpins; PHs). These putative PH-stabilized pause signals were enriched in 5′ untranslated regions and protein coding regions in the E. coli genome. These results suggest that the SI3 domain affects different classes of pause signals differently and imply potential roles of these pauses in protein-coding regions.

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