The quaternary geometry of transcription termination factor rho: assignment by chemical cross-linking

Horiguchi, Taigo; Miwa, Yoshiro; Shigesada, Katsuya

doi:10.1006/jmbi.1997.1059

Cited by 31 publications

(35 citation statements)

References 38 publications

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“…A similar situation has been proposed for Rho (19,47,52); in a type of tethered tracking model for termination (53,54), tight binding sites may hold RNA relatively immobile around the outside of the Rho hexamer, whereas ATP hydrolysis activity is coordinated with looser RNA binding and release in central sites. The net result is feeding of the 3Ј end of the RNA through the center of Rho as the protein moves 5Ј to 3Ј along the RNA (19,52). This type of model could provide the asymmetry required by our present results.…”

Section: Sequential Hydrolysis Of 3 Atp Molecules Bound To Rho Requirsupporting

confidence: 53%

“…In F 1 there is rotation of the central ␥ subunit within the ␣ 3 ␤ 3 hexamer (48,49), and for T7 4AЈ helicase DNA binding is to one or two subunits of the hexamer within the central region (50,51). A similar situation has been proposed for Rho (19,47,52); in a type of tethered tracking model for termination (53,54), tight binding sites may hold RNA relatively immobile around the outside of the Rho hexamer, whereas ATP hydrolysis activity is coordinated with looser RNA binding and release in central sites. The net result is feeding of the 3Ј end of the RNA through the center of Rho as the protein moves 5Ј to 3Ј along the RNA (19,52).…”

Section: Sequential Hydrolysis Of 3 Atp Molecules Bound To Rho Requirmentioning

confidence: 52%

“…An additional class of three ATP-binding sites of lower affinity has also been suggested (16), although the catalytic activity of these sites was not assessed. The stoichiometry for ATP and RNA oligomer interactions with Rho, together with studies of Rho quaternary structure (17)(18)(19), have led to a proposed model of Rho as a trimer of dimers in which adjacent identical subunits may alternate in their ability to bind and hydrolyze ATP and to bind and release RNA (8,15,20).Our goal is to elucidate the molecular mechanism of Rho activity, including the sequence of ATP and RNA binding, ATP hydrolysis, product release, and interactions with other molecules that sum to transcription termination. Travel in a 5Ј 3 3Ј direction along single-stranded RNA can readily be seen as consistent with the hydrolysis in an ordered sequence of ATP molecules that are bound to Rho, but evidence for such a hydrolysis pattern is lacking.…”

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Sequential Hydrolysis of ATP Molecules Bound in Interacting Catalytic Sites of Escherichia coli Transcription Termination Protein Rho

Stitt

1998

Journal of Biological Chemistry

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Escherichia coli transcription termination protein Rho aids in the release of newly synthesized RNA from paused transcription complexes (reviewed in Ref. 1). The homohexameric protein binds nascent RNA and, with the RNA-dependent hydrolysis of ATP, disrupts the ternary transcription complex, releasing product RNA and allowing RNA polymerase to recycle. The discovery of a 5Ј 3 3Ј RNA-DNA helicase activity of Rho (2) suggested that Rho might disrupt the RNA-DNA duplex of the transcription bubble. Recent studies of ternary transcription complexes (Refs. 3-5 and reviewed in Ref. 6) suggest that such disruption could be important in transcription termination, as could be the release of the nascent RNA just 5Ј of the RNA-DNA duplex from its interactions with RNA polymerase. As described by Nudler et al. (7), the interaction of RNA with RNA polymerase immediately 5Ј from the RNA-DNA hybrid may control the opening and closing of an RNA polymerase clamp around the DNA template near the leading edge of the enzyme, and contribute to the stability of the ternary transcription complex. An appealing model for Rho is one in which the enzyme binds to exposed mRNA behind RNA polymerase and travels 5Ј 3 3Ј along the RNA as it hydrolyzes ATP, binding and releasing RNA from different parts of the hexamer to accomplish movement (8). Such activity could release nascent RNA from RNA polymerase-binding sites and could constitute the basis for its RNA-DNA helicase activity, both of which might be involved in transcript release from paused ternary transcription complexes. The finding that the same number of ATP molecules per RNA length is hydrolyzed by Rho traveling along RNA and Rho unwinding RNA-DNA hybrids (8) supports this hypothesis.Rho binds single-stranded RNA, showing preferred entry regions on RNA upstream of eventual transcription termination sites. However, the characteristics of these regions, beyond low secondary structure and some preference for a C-rich, G-poor base composition (9), are too poorly understood to permit their identification by sequence inspection. When bound to RNA, Rho protects 80 bases from ribonuclease degradation (10, 11). The binding of Rho to 10-base RNA oligomers was reported as best fit by three tight and three weaker sites per hexamer (12, 13).The RNA-dependent hydrolysis of ATP is essential for the transcription termination function of Rho. Two components of ternary transcription complexes, the DNA template and RNA polymerase, are not required to elicit this ATPase activity, thus considerably simplifying study of the reaction (14). The reaction is particularly well stimulated by the RNA homopolymer poly(C), and Rho is frequently assayed in vitro by measuring its poly(C)-dependent ATPase activity. Previous work has shown that the Rho hexamer binds three molecules of MgATP in a single class of catalytically competent sites (15,16). An additional class of three ATP-binding sites of lower affinity has also been suggested (16), although the catalytic activity of these sites was not assessed. The stoic...

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Section: Sequential Hydrolysis Of 3 Atp Molecules Bound To Rho Requirsupporting

confidence: 53%

Section: Sequential Hydrolysis Of 3 Atp Molecules Bound To Rho Requirmentioning

confidence: 52%

mentioning

confidence: 99%

mentioning

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Sequential Hydrolysis of ATP Molecules Bound in Interacting Catalytic Sites of Escherichia coli Transcription Termination Protein Rho

Stitt

1998

Journal of Biological Chemistry

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“…The primary site of action in Escherichia coli is unique and has been identified as the transcription termination factor, Rho (9). Rho is composed of six identical 47-kDa proteins of 419 amino acids (10) and exists in a planar, hexagonal (11)(12)(13) arrangement of proposed C 6 (14) or D 3 (15) geometry. Rho-dependent transcription termination sites occur at the ends of transcription units, at regulatory points before or between genes, and within genes (16).…”

Section: Bicyclomycin (Bcm)mentioning

confidence: 99%

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

Riba¹,

Gaskell²,

Cho

et al. 1998

Journal of Biological Chemistry

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The commercial antibiotic bicyclomycin (Bcm) has been shown to target the essential transcription termination factor Rho in Escherichia coli. Little is known about the Bcm binding domain in Rho. A recent structure-activity relationship study led us to evaluate the reductive amination probe, 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm). Biochemical studies showed that FD-Bcm possessed inhibitory activities comparable to Bcm in Rho-dependent ATPase and transcription termination assays. Incubation of Rho with FD-Bcm, ATP, and poly(C) followed by NaBH 4 reduction and dialysis led to an appreciable loss of ATPase activity. Inclusion of Bcm with FD-Bcm in the reductive amination reaction protected Rho, indicating that Bcm and FD-Bcm competed for the same binding site in Rho. Incubation of Rho with FD-Bcm and poly(C) followed by NaBH 4 reduction provided a sample with residual ATPase activity (12%). Mass spectrometric analysis indicated the presence of two proteins in an approximate 1.2:1 ratio, whose masses corresponded to wild-type Rho (47,010 Da) and lysine-modified Rho (47,417 Da), respectively. Trypsin digestion of the Rho sample followed by high performance liquid chromatography separation and tandem mass spectrometry analysis identified the site of modification as Lys 181 within the combined tryptic fragment, Gly-Leu-Ile-Val-Ala-Pro-Pro-Lys-Ala-Gly-Lys (residues 174 -184). Similar analysis of a lesser modified sample (following incubation with inclusion of ATP) showed that addition had again occurred at Lys 181 . These findings provide the first structural information concerning the site of Bcm binding in Rho.Bicyclomycin (Bcm) 1 is a commercial antibiotic of novel structure (1-8). The primary site of action in Escherichia coli is unique and has been identified as the transcription termination factor, Rho (9). Rho is composed of six identical 47-kDa proteins of 419 amino acids (10) and exists in a planar, hexagonal (11-13) arrangement of proposed C 6 (14) or D 3 (15) geometry. Rho-dependent transcription termination sites occur at the ends of transcription units, at regulatory points before or between genes, and within genes (16). Transcription termination begins with the recognition and binding of Rho to discrete regions (rut sites) within the newly synthesized RNA chains (17). Binding is governed by a general requirement for a cytosine-rich sequence, and it extends across all six subunits of the functional protein, encompassing approximately 80 nucleotides (17)(18)(19)(20). Rho then extends its interaction toward the 3Ј end of the RNA polymerase elongation complex in a process coupled to ATP hydrolysis. The Rho-mediated release of RNA from the transcription bubble results from a destabilization of the complex, believed to be caused, in part, by the ATPase-dependent 5Ј 3 3Ј RNA-DNA helicase action of the protein (21).Details of the Bcm-Rho interaction have emerged in recent investigations (9,(22)(23)(24). Chemical studies have shown that nucleophilic amino acids readily react with Bcm, consistent with sever...

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Polymorphic quaternary organization of the Bacillus subtilis bacteriophage SPP1 replicative helicase (G 40 P) 1 1Edited by W. Baumeister

Bárcena

Martı́n

Weise

et al. 1998

Journal of Molecular Biology

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The quaternary geometry of transcription termination factor rho: assignment by chemical cross-linking

Cited by 31 publications

References 38 publications

Sequential Hydrolysis of ATP Molecules Bound in Interacting Catalytic Sites of Escherichia coli Transcription Termination Protein Rho

Sequential Hydrolysis of ATP Molecules Bound in Interacting Catalytic Sites of Escherichia coli Transcription Termination Protein Rho

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

Polymorphic quaternary organization of the Bacillus subtilis bacteriophage SPP1 replicative helicase (G 40 P) 1 1Edited by W. Baumeister

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