A Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho

ABSTRACT RfaH activates horizontally acquired operons that encode lipopolysaccharide core components, pili, toxins, and capsules. Unlike its paralog NusG, which potentiates Rho-mediated silencing, RfaH strongly inhibits Rho. RfaH is recruited to its target operons via a network of contacts with an e...

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Autores principales:	Kuang Hu, Irina Artsimovitch
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	RNA polymerase RfaH Rho polarity termination Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/233c1f8d717641babaa2a9ceb4255fa6
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spelling	oai:doaj.org-article:233c1f8d717641babaa2a9ceb4255fa62021-11-15T15:51:29ZA Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho10.1128/mBio.00753-172150-7511https://doaj.org/article/233c1f8d717641babaa2a9ceb4255fa62017-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00753-17https://doaj.org/toc/2150-7511ABSTRACT RfaH activates horizontally acquired operons that encode lipopolysaccharide core components, pili, toxins, and capsules. Unlike its paralog NusG, which potentiates Rho-mediated silencing, RfaH strongly inhibits Rho. RfaH is recruited to its target operons via a network of contacts with an elongating RNA polymerase (RNAP) and a specific DNA element called ops to modify RNAP into a pause- and NusG-resistant state. rfaH null mutations confer hypersensitivity to antibiotics and detergents, altered susceptibility to bacteriophages, and defects in virulence. Here, we carried out a selection for suppressors that restore the ability of a ΔrfaH mutant Escherichia coli strain to grow in the presence of sodium dodecyl sulfate. We isolated rho, rpoC, and hns suppressor mutants with changes in regions previously shown to be important for their function. In addition, we identified mutants with changes in an unstructured region that connects the primary RNA-binding and helicase domains of Rho. The connector mutants display strong defects in vivo, consistent with their ability to compensate for the loss of RfaH, and act synergistically with bicyclomycin (BCM), which has been recently shown to inhibit Rho transformation into a translocation-competent state. We hypothesize that the flexible connector permits the reorientation of Rho domains and serves as a target for factors that control the motor function of Rho allosterically. Our results, together with the existing data, support a model in which the connector segment plays a hitherto overlooked role in the regulation of Rho-dependent termination. IMPORTANCE The transcription termination factor Rho silences foreign DNA, reduces antisense transcription, mediates surveillance of mRNA quality, and maintains genome integrity by resolving transcription-replication collisions and deleterious R loops. Upon binding to RNA, Rho undergoes a rate-limiting transition from an open “lock washer” state to a closed ring capable of processive translocation on, and eventually the release of, the nascent transcript. Recent studies revealed that Rho ligands, including its cofactor NusG and inhibitor bicyclomycin, control the ring dynamics allosterically. In this work, we used a genetic selection for suppressors of RfaH, a potent inhibitor of Rho, to isolate a new class of mutations in a flexible region that connects the primary RNA-binding and ATPase/translocase domains of Rho. We propose that the connector is essential for the modulation of Rho activity by different RNA sequences and accessory proteins.Kuang HuIrina ArtsimovitchAmerican Society for MicrobiologyarticleRNA polymeraseRfaHRhopolarityterminationMicrobiologyQR1-502ENmBio, Vol 8, Iss 3 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	RNA polymerase RfaH Rho polarity termination Microbiology QR1-502
spellingShingle	RNA polymerase RfaH Rho polarity termination Microbiology QR1-502 Kuang Hu Irina Artsimovitch A Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho
description	ABSTRACT RfaH activates horizontally acquired operons that encode lipopolysaccharide core components, pili, toxins, and capsules. Unlike its paralog NusG, which potentiates Rho-mediated silencing, RfaH strongly inhibits Rho. RfaH is recruited to its target operons via a network of contacts with an elongating RNA polymerase (RNAP) and a specific DNA element called ops to modify RNAP into a pause- and NusG-resistant state. rfaH null mutations confer hypersensitivity to antibiotics and detergents, altered susceptibility to bacteriophages, and defects in virulence. Here, we carried out a selection for suppressors that restore the ability of a ΔrfaH mutant Escherichia coli strain to grow in the presence of sodium dodecyl sulfate. We isolated rho, rpoC, and hns suppressor mutants with changes in regions previously shown to be important for their function. In addition, we identified mutants with changes in an unstructured region that connects the primary RNA-binding and helicase domains of Rho. The connector mutants display strong defects in vivo, consistent with their ability to compensate for the loss of RfaH, and act synergistically with bicyclomycin (BCM), which has been recently shown to inhibit Rho transformation into a translocation-competent state. We hypothesize that the flexible connector permits the reorientation of Rho domains and serves as a target for factors that control the motor function of Rho allosterically. Our results, together with the existing data, support a model in which the connector segment plays a hitherto overlooked role in the regulation of Rho-dependent termination. IMPORTANCE The transcription termination factor Rho silences foreign DNA, reduces antisense transcription, mediates surveillance of mRNA quality, and maintains genome integrity by resolving transcription-replication collisions and deleterious R loops. Upon binding to RNA, Rho undergoes a rate-limiting transition from an open “lock washer” state to a closed ring capable of processive translocation on, and eventually the release of, the nascent transcript. Recent studies revealed that Rho ligands, including its cofactor NusG and inhibitor bicyclomycin, control the ring dynamics allosterically. In this work, we used a genetic selection for suppressors of RfaH, a potent inhibitor of Rho, to isolate a new class of mutations in a flexible region that connects the primary RNA-binding and ATPase/translocase domains of Rho. We propose that the connector is essential for the modulation of Rho activity by different RNA sequences and accessory proteins.
format	article
author	Kuang Hu Irina Artsimovitch
author_facet	Kuang Hu Irina Artsimovitch
author_sort	Kuang Hu
title	A Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho
title_short	A Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho
title_full	A Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho
title_fullStr	A Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho
title_full_unstemmed	A Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho
title_sort	screen for <italic toggle="yes">rfah</italic> suppressors reveals a key role for a connector region of termination factor rho
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/233c1f8d717641babaa2a9ceb4255fa6
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A Screen for <italic toggle="yes">rfaH</italic> Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho

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