Influence of Substrate Composition on the Helicase Activity of Transcription Termination Factor Rho: Reduced Processivity of Rho Hexamers during Unwinding of RNA–DNA Hybrid Regions

Walmacq, Céline; Rahmouni, A. Rachid; Boudvillain, Marc

doi:10.1016/j.jmb.2004.07.026

Cited by 29 publications

(104 citation statements)

References 64 publications

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“…5C). This confirmed that the rut sequence rich in C residues was needed for efficient Rho-dependent termination, a finding that was in keeping with descriptions of other Rho-dependent terminators (2,14,17,20,26,33,39,46,60,62).…”

Section: Resultssupporting

confidence: 84%

“…Rho-dependent terminators lack the obvious structural features that are associated with intrinsic terminators (7). In general, they consist of a bipartite structure that extends for up to 150 bp of DNA and have a high proportion of C relative to G residues (2,14,20,33,39,44,60,62). The Rho utilization (rut) site of a Rho-dependent terminator is a site on the RNA transcript which generally has little secondary structure and is recognized by the RNAbinding Rho factor, while the transcription stop point (tsp) is the region within which Rho enhances transcription termination of paused RNA polymerase complexes (6,17,35,45).…”

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confidence: 99%

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Characterization of the Detachable Rho-Dependent Transcription Terminator of the fimE Gene in Escherichia coli K-12

2005

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The fim genetic switch in the chromosome of Escherichia coli K-12 is an invertible DNA element that harbors the promoter for transcription of the downstream fim structural genes and a transcription terminator that acts on the upstream fimE regulatory gene. Switches oriented appropriately for structural gene transcription also allow fimE mRNA to read through, whereas those in the opposite orientation terminate the fimE message. We show here that termination is Rho dependent and is suppressed in a rho mutant or by bicyclomycin treatment when fimE mRNA is expressed by the fimE gene, either from a multicopy recombinant plasmid or in its native chromosomal location. Two cis-acting elements within the central portion of the 314-bp invertible DNA switch were identified as contributors to Rho-dependent termination and dissected. These fim sequence elements show similarities to well-characterized Rho utilization (rut) sites and consist of a boxA motif and a C-rich and G-poor region of approximately 40 bp. Deletion of the boxA motif alone had only a subtle negative effect on Rho function. However, when this element was deleted in combination with the C-rich, G-poor region, Rho function was considerably decreased. Altering the C-to-G ratio in favor of G in this portion of the switch also strongly attenuated transcription termination. The implications of the existence of a fimE-specific Rho-dependent terminator within the invertible switch are discussed in the context of the fim regulatory circuit.Escherichia coli and other bacteria end the process of transcript elongation by employing one of the following two types of transcription terminator: intrinsic terminators and factor-dependent terminators (36,40,43,58). Intrinsic terminators typically consist of an approximately 20-bp stretch of DNA that is composed of a GϩC region of hyphenated dyad symmetry followed by a run of T residues. This specifies in the RNA transcript a stable stem-loop structure that is followed by up to eight U residues (8, 31, 50). These U residues base pair weakly with the corresponding A residues in the template DNA strand, and in combination with the formation of the RNA stem-loop structure, lead to destabilization of the RNA polymerase-template-transcript elongation complex and the consequent termination event (43, 61).Factor-dependent terminators utilize the hexameric ringshaped Rho protein to facilitate transcript release from regions of the template that are not characterized by intrinsic RNA-DNA hybrid helix instability (5,7,37,40,47). Rho-dependent terminators lack the obvious structural features that are associated with intrinsic terminators (7). In general, they consist of a bipartite structure that extends for up to 150 bp of DNA and have a high proportion of C relative to G residues (2,14,20,33,39,44,60,62). The Rho utilization (rut) site of a Rho-dependent terminator is a site on the RNA transcript which generally has little secondary structure and is recognized by the RNAbinding Rho factor, while the transcription stop point (tsp) is th...

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Section: Resultssupporting

confidence: 84%

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confidence: 99%

Characterization of the Detachable Rho-Dependent Transcription Terminator of the fimE Gene in Escherichia coli K-12

2005

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“…18 Interestingly, the T7 ring helicase also displays a reduced processivity during DNA unwinding. 30 Isolated P4 proteins exhibit marked differences in their translocation processivity, depending on the stability of their rings.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

“…However, ec Rho unwinds RNA-DNA helices with a surprisingly moderate processivity of ~0.985 per unwound base pair (bp). 18 This indicates that, on average, the ec Rho helicase cannot unwind more than 60-70 bp of RNA-DNA hybrid before dissociating from its substrate. Thus, ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

“…The transcription termination factor Rho from E. coli ( ec Rho) unwinds RNA and RNA-DNA helices in a NTP-dependent fashion, [16][17][18][19][20][21][22][23] a property that is likely connected to Rho-induced dissociation of transcription elongation complexes (TECs; composed of the DNA template, RNA polymerase and transcript; Fig. 2B) at specific loci of bacterial genomes, termed Rho-dependent terminators.…”

Section: Introductionmentioning

confidence: 99%

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RNA remodeling by hexameric RNA helicases

Rabhi¹,

Tůma²,

Boudvillain³

2010

RNA Biology

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A Simple Fluorescence Microplate Assay to Monitor RNA-DNA Hybrid Unwinding by the Bacterial Transcription Termination Factor Rho

Simón

Boudvillain

2020

Methods in Molecular Biology

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Transcription termination factor Rho contributes to shape the transcriptomes of many bacteria and is essential in a large subset of them. Although the transcription termination function of Rho is not always easy to reconstitute and to study in vitro, assays based on the ATPdependent RNA-DNA hybrid unwinding activity of the factor can prove useful to dissect Rho mechanisms or to seek new antibiotics targeting Rho. However, current in vitro assays of Rho helicase activity are time-consuming, as they usually require radiolabeling of the hybrid substrates and analysis of reaction products by gel electrophoresis. Here, we describe a fluorescence-based microplate assay that informs on Rho helicase activity in a matter of minutes and allows the multiplexed analysis of conditions required for primary biochemical characterization or for drug screening.

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Influence of Substrate Composition on the Helicase Activity of Transcription Termination Factor Rho: Reduced Processivity of Rho Hexamers during Unwinding of RNA–DNA Hybrid Regions

Cited by 29 publications

References 64 publications

Characterization of the Detachable Rho-Dependent Transcription Terminator of the fimE Gene in Escherichia coli K-12

Characterization of the Detachable Rho-Dependent Transcription Terminator of the fimE Gene in Escherichia coli K-12

RNA remodeling by hexameric RNA helicases

A Simple Fluorescence Microplate Assay to Monitor RNA-DNA Hybrid Unwinding by the Bacterial Transcription Termination Factor Rho

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