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Allosteric couplings upon binding of RfaH to transcription elongation complexes.

ABSTRACT: In every domain of life, NusG-like proteins bind to the elongating RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes. Structures of factor-bound transcription elongation complexes (TECs) reveal similar contacts to RNAP, consistent with a shared mechanism of action. However, NusG homologs differ in their regulatory roles, modes of recruitment, and effects on RNA synthesis. Some of these differences could be due to conformational changes in RNAP and NusG-like proteins, which cannot be captured in static structures. Here, we employed hydrogen-deuterium exchange mass spectrometry to investigate changes in local and non-local structural dynamics of Escherichia coli NusG and its paralog RfaH, which have opposite effects on expression of xenogenes, upon binding to TEC. We found that NusG and RfaH regions that bind RNAP became solvent-protected in factor-bound TECs, whereas RNAP regions that interact with both factors showed opposite deuterium uptake changes when bound to NusG or RfaH. Additional changes far from the factor-binding site were observed only with RfaH. Our results provide insights into differences in structural dynamics exerted by NusG and RfaH during binding to TEC, which may explain their different functional outcomes and allosteric regulation of transcriptional pausing by RfaH.

SUBMITTER: Molina JA

PROVIDER: S-EPMC9226497 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nucleoid-associated proteins (NAPs) silence xenogenes by blocking RNA polymerase binding to promoters and hindering transcript elongation. In Escherichia coli, H-NS and its homolog SptA interact with YmoA proteins Hha and YdgT to assemble nucleoprotein filaments that facilitate transcription termination by Rho, which acts in synergy with NusG. Countersilencing during initiation is facilitated by proteins that exclude NAPs from promoter regions, but auxiliary factors that alleviate silencing during elongation are not known. A specialized NusG paralog, RfaH, activates lipopolysaccharide core biosynthesis operons, enabling survival in the presence of detergents and antibiotics. RfaH strongly inhibits Rho-dependent termination by reducing RNA polymerase pausing, promoting translation, and competing with NusG. We hypothesize that RfaH also acts as a countersilencer of NAP/YmoA filaments. We show that deletions of hns and hha+ydgT suppress the growth defects of ΔrfaH by alleviating Rho-mediated polarity within the waa operon. The absence of YmoA proteins exacerbates cellular defects caused by reduced Rho levels or Rho inhibition by bicyclomycin but has negligible effects at a strong model Rho-dependent terminator. Our findings that the distribution of Hha and RfaH homologs is strongly correlated supports a model in which they comprise a silencing/countersilencing pair that controls expression of chromosomal and plasmid-encoded xenogenes. IMPORTANCE Horizontally acquired DNA drives bacterial evolution, but its unregulated expression may harm the recipient. Xenogeneic silencers recognize foreign genes and inhibit their transcription. However, some xenogenes, such as those encoding lipo- and exopolysaccharides, confer resistance to antibiotics, bile salts, and detergents, necessitating the existence of countersilencing fitness mechanisms. Here, we present evidence that Escherichia coli antiterminator RfaH alleviates silencing of the chromosomal waa operon and propose that plasmid-encoded RfaH homologs promote dissemination of antibiotic resistance genes through conjugation.

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Allosteric couplings upon binding of RfaH to transcription elongation complexes.

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