Transcription termination factor rho is an RNA-DNA helicase

Brennan, Catherine A.; Dombroski, Alicia J.; Platt, Terry

doi:10.1016/0092-8674(87)90703-3

Cited by 236 publications

(181 citation statements)

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“…Rho Exhibits ATP-dependent RNA-DNA Helicase Activity on a Circular RNA Substrate-Rho can utilize the energy derived from ATP hydrolysis to translocate along a RNA in a 3Ј direction and to disrupt RNA-DNA hybrids that are 3Ј of the primary Rho attachment site (3,4). To determine if Rho can perform this ATP-driven reaction on a circular RNA, a DNA oligonucleotide complementary to a sequence on the 3Ј side of the rut site (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Rho also can dissociate a RNA molecule bound to DNA through a short hybrid helix in a reaction that is dependent on ATP hydrolysis and the presence of an attachment site for Rho on the RNA on the 5Ј side of the hybrid helix (3)(4)(5). This RNA-DNA helicase activity may mimic the process of removal of the nascent RNA strand from the transcription elongation complex.…”

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confidence: 99%

“…Rho will disrupt RNA-DNA hybrids located 3Ј of the rut site but not hybrids located at the 5Ј-end of the transcript (3,4). Another way to demonstrate Rho's ability to act on a circular RNA is to show that it can disrupt a hybrid with a DNA oligonucleotide (called 5Ј oligonucleotide) that is complementary to a segment of RNA near the 5Ј-end of the original transcript that was used to make the circular derivative (Fig.…”

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Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Burgess

Richardson

2001

Journal of Biological Chemistry

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Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5 terminus. To determine whether Rho's activity is 5-end-dependent, its ability to bind to and function on a circular derivative of cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of cro RNA containing an extra 10-nucleotide sequence near the 5-end that was complementary to a sequence located near the 3-end. Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5 terminus.Transcription termination factor Rho is an RNA-binding protein that couples the energy derived from ATP hydrolysis to actions that dissociate a RNA transcript from its biosynthetic complex with DNA and RNA polymerase (1, 2). Rho also can dissociate a RNA molecule bound to DNA through a short hybrid helix in a reaction that is dependent on ATP hydrolysis and the presence of an attachment site for Rho on the RNA on the 5Ј side of the hybrid helix (3-5). This RNA-DNA helicase activity may mimic the process of removal of the nascent RNA strand from the transcription elongation complex.Rho factor is believed to function in vivo as a hexamer consisting of six polypeptide subunits arranged in a ring-shaped structure (6, 7). Burgess and Richardson (8) have recently presented a model for the Rho-RNA complex in which a segment of RNA, called a rut site (Rho utilization site), binds to a cleft comprised of the N-terminal RNA-binding domains of the six individual subunits located at one end of the hexameric structure (the crown) (9 -11). RNA sequence 3Ј of the rut site then passes into the hole located at the center of the hexamer. Evidence for passage of a single-stranded nucleic acid substrate through a ring-shaped hexameric structure has been observed for other hexameric helicases, such as DnaB and the T7 gene 4 product (12, 13).The process by which Rho binds to and captures the 3Ј-end of an RNA in the center of the hexamer is not known. Gan and Richardson (14) have recently presented data indicating that Rho, in vitro, could form hexamers by partial assembly of subunits on an RNA. Another mechanism could have the RNA enter into the center of the hexameric structure through an opening or notch in the ring (7). A third mechanism may involve Rho threading onto the nascent transcript from the free 5Ј-end of the RNA. To test the model in which Rho requires a free end of RNA to thread onto in order to function as a terminator, we investigated Rho's ability to utilize its ATP-dependent helicase activity on an RNA lac...

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Section: Resultsmentioning

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Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Burgess

Richardson

2001

Journal of Biological Chemistry

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“…This ATPase activity and its cofactor requirements have been extensively studied (Lowery & Richardson, 1977a,b;Galluppi & Richardson, 1980;McSwiggen, 1985;von Hippel et al, 1987;Faus & Richardson, 1989). Using this RNA-dependent ATPase as an energy source, Brennan et al (1987) have shown that rho can act as a 5' 4 3' directional RNA-DNA helicase on RNA-DNA heteroduplex constructs that carry a singlestranded region of RNA at the 5' end. This helicase action is thought to be central to the process of transcript release at rho-dependent termination sites.…”

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Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA

1992

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The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC], where n 2 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds -70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC), length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo(rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho. Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.

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“…Termination in Escherichia coli is largely mediated by Rho, an RNAdependent ATPase that terminates transcription promoter-distal to unstructured and untranslated RNA (12,13). The RNA/DNA helicase activity of Rho promotes termination by unwinding RNA/DNA hybrid in the RNAP transcription bubble (14). Inhibition of Rho with bicyclomycin (BCM) or deletion of the Rho accessory factors, NusA and NusG, massively disregulate gene expression in E. coli (15).…”

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Transcription termination maintains chromosome integrity

Washburn

Gottesman²

2010

Proc. Natl. Acad. Sci. U.S.A.

108

152

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DNA replication fork movement is impeded by collisions with transcription elongation complexes (TEC). We propose that a critical function of transcription termination factors is to prevent TEC from blocking DNA replication and inducing replication fork arrest, one consequence of which is DNA double-strand breaks. We show that inhibition of Rho-dependent transcription termination by bicyclomycin in Escherichia coli induced double-strand breaks. Cells deleted for Rho-cofactors nusA and nusG were hypersensitive to bicyclomycin, and had extensive chromosome fragmentation even in the absence of the drug. An RNA polymerase mutation that destabilizes TEC (rpoB*35) increased bicyclomycin resistance >40-fold. Double-strand break formation depended on DNA replication, and can be explained by replication fork collapse. Deleting recombination genes required for replication fork repair (recB and ruvC) increased sensitivity to bicyclomycin, as did loss of the replication fork reloading helicases rep and priA. We propose that Rho responds to a translocating replisome by releasing obstructing TEC.DNA polymerase/RNA polymerase collisions | pulsed-field gel electrophoresis | SOS D NA replication forks translocate 20 to 30 times faster (∼600 nt/s) than the rate of RNA polymerase (RNAP) elongation (∼20 nt/s) (1-3). This finding raises the possibility of repeated collisions between replication forks and transcription elongation complexes (TEC), even when DNA and RNA synthesis are codirectional. Collisions between replication forks and TEC can generate fork collapse and chromosomal double-strand breaks (DSBs) (4, 5). Mechanisms have evolved, therefore, to reduce collisions and to repair collapsed forks. Fork repair is promoted by multiple pathways that involve a variety of DNA repair functions (6). Recombination can restart replisomes blocked by arrested TEC (7,8). Boubakri et al. (9) have demonstrated that DNA helicases Rep, DinG, and UvrD resolve head-on collisions between TEC in rrn operons and replisomes. A recent report describes the role of transcription elongation factors DksA and GreA in preventing conflicts between replication and transcription (7, 10). In vitro, replication forks that collapse after colliding with a codirectional TEC can restart, using an R-loop (RNA-DNA hybrid) from a transcription bubble as primer (11), although it is not known if this occurs in vivo.Transcription termination isolates transcription units and prevents inappropriate expression of downstream genes. Termination in Escherichia coli is largely mediated by Rho, an RNAdependent ATPase that terminates transcription promoter-distal to unstructured and untranslated RNA (12, 13). The RNA/DNA helicase activity of Rho promotes termination by unwinding RNA/DNA hybrid in the RNAP transcription bubble (14). Inhibition of Rho with bicyclomycin (BCM) or deletion of the Rho accessory factors, NusA and NusG, massively disregulate gene expression in E. coli (15). Surprisingly, efficient transcription termination is only required to suppress expression of cryp...

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Transcription termination factor rho is an RNA-DNA helicase

Cited by 236 publications

References 34 publications

Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA

Transcription termination maintains chromosome integrity

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