Structural insights into RNA-mediated transcription regulation in bacteria

Dey, Sanjay; Batisse, Claire; Shukla, Jinal K.; Webster, M.; Takács, Mária; Saint-André, Charlotte; Weixlbaumer, A.

doi:10.1016/j.molcel.2022.09.020

Cited by 15 publications

(8 citation statements)

References 84 publications

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“…Additionally, ternary interactions not captured in our prediction may stabilize PHs ( 67 ). Further analysis of the ΔSI3-suppressed pause sites using improved algorithms for RNA structure prediction that can detect complex RNA structures involving more nascent RNA may reveal additional hs-pause sites that tune transcription via novel mechanisms ( 68 , 69 ).…”

Section: Discussionmentioning

confidence: 99%

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

Bao,

Cao,

Landick

2024

Nucleic Acids Research

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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.

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Section: Discussionmentioning

confidence: 99%

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

Bao,

Cao,

Landick

2024

Nucleic Acids Research

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“…On the one hand, interactions with nucleotides, RNAs, or NusG shift the equilibrium between open and closed hexameric states and thereby regulate ρ ATPase and RNA translocase activities. 22,56 On the other hand, RNAP-associated NusA 57,58 , an RNAP-trailing ribosome [59][60][61] , RNAP-bound elements of nascent RNA 14,15 , RNAP-bound specialized transcription factors [11][12][13] , or multi-factorial RNAP-bound RNA-protein complexes 9,10 can delay or prevent the attack of ECs by ρ. Furthermore, the RNA chaperone Hfq can counteract ρ by depositing ρ-inhibitory sRNAs on nascent transcripts.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, phages that utilize the bacterial transcription machinery must counteract ρ activity to efficiently express their genomes. While lambdoid phages implement protein-or RNA-based antitermination mechanisms to render the host RNA polymerase (RNAP) termination-resistant [9][10][11][12][13][14][15] , the enterobacterial pirate phage, P4, has acquired a unique anti-termination system that acts directly upon ρ. Its polarity suppression (Psu) protein is a building block of its capsid 16 , but can also inhibit ρ 17,18 , as first noted by its suppression of ρ-dependent polarity 19 .…”

Section: Mainmentioning

confidence: 99%

Widespread gene regulator Psu inhibits transcription termination factor ρ by forced hyper-oligomerization

Gjorgjevikj

Kumar

Wang

et al. 2023

Preprint

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Many bacteriophages modulate the host transcription machinery for efficient expression of their own genomes. Phage P4 polarity suppression protein, Psu, is a building block of the viral capsid and inhibits the hexameric transcription termination factor, rho by presently unknown mechanisms. We elucidated cryogenic electron microscopy structures of rho-Psu complexes, showing that Psu dimers laterally clamp two inactive, open rho rings and promote their expansion to higher-oligomeric states. Systematic ATPase, nucleotide binding and nucleic acid binding studies revealed that Psu hinders rho ring closure and traps nucleotides in their binding pockets on rho. Structure-guided mutagenesis in combination with growth, pull-down and termination assays further delineated the functional rho-Psu interfaces. Bioinformatic analyses suggested that, in addition to guarding its own genome against rho, Psu enables expression of diverse phage-defense systems commonly found in P4-like mobile genetic elements across bacteria. Thus, Psu is a widespread gene regulator that inhibits rho via forced hyper-oligomerization.

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“…Bacteriophages, pervasive mobile elements that are silenced by ρ 8 , 9 , use analogous mechanisms to ensure that their genes are expressed. Anti-termination is mediated by large ribonucleoprotein complexes, e.g., during rRNA synthesis or phage λ gene expression 10 , 11 , single proteins such as RfaH 12 , 13 or RNAs 14 – 16 , which are recruited to specific sequences or encoded within their target operons and inhibit termination within just one or a few operons.…”

Section: Introductionmentioning

confidence: 99%

Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation

Said,

Finazzo,

Hilal

et al. 2024

Nat Commun

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Transcription termination factor ρ is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits ρ-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Here, our cryo-EM structures of ρ-Rof and ρ-RNA complexes show that Rof undergoes pronounced conformational changes to bind ρ at the protomer interfaces, undercutting ρ conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between ρ and RNA polymerase. Consistently, Rof impedes ρ ring closure, ρ-RNA interactions and ρ association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirms that the observed ρ-Rof interface is required for Rof-mediated inhibition of cell growth and ρ-termination in vitro. Bioinformatic analyses reveal that Rof is restricted to Pseudomonadota and that the ρ-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence ρ under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by ρ may be detrimental.

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Structural insights into RNA-mediated transcription regulation in bacteria

Cited by 15 publications

References 84 publications

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

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

Widespread gene regulator Psu inhibits transcription termination factor ρ by forced hyper-oligomerization

Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation

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