Structure and assembly of the Escherichia coli transcription termination factor rho and its interactions with RNA I. Cryoelectron microscopic studies

Gogol, Edward P.; Seifried, Steven E.; Hippel, Peter H. von

doi:10.1016/0022-2836(91)90923-t

Cited by 97 publications

(99 citation statements)

References 21 publications

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“…This disparity could be the result of differences in imaging methods, nucleotide, and DNA used, as well as other factors. However, averaged cryoelectron microscopic images of another helicase, the E. coli rho protein, clearly show both closed and ''notched'' rings very similar to those presented here (15). Moreover, a model has been proposed based on both hydrody- namic and electron microscopic data (9) in which assembly of the T4 helicase around DNA is a process in which ''open hexamers'' are formed before ''closed hexamers.''…”

Section: Resultssupporting

confidence: 70%

Architecture of the bacteriophage T4 primosome: Electron microscopy studies of helicase (gp41) and primase (gp61)

Norcum

Warrington

Spiering

et al. 2005

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

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Replication of DNA requires helicase and primase activities as part of a primosome assembly. In bacteriophage T4, helicase and primase are separate polypeptides for which little structural information is available and whose mechanism of association within the primosome is not yet understood. Three-dimensional structural information is provided here by means of reconstructions from electron microscopic images. Structures have been calculated for complexes of each of these proteins with ssDNA in the presence of MgATP␥S. Both the helicase (gp41) and primase (gp61) complexes are asymmetric hexagonal rings. The gp41 structure suggests two distinct forms that have been termed ''open'' and ''closed.'' The gp61 structure is clearly a six-membered ring, which may be a trimer of dimers or a traditional hexamer of monomers. This structure provides conclusive evidence for an oligomeric primaseto-ssDNA stoichiometry of 6:1.three-dimensional structure ͉ DNA replication ͉ protein hexamers ͉ replisome T he replicative helicases and primases are members of the primosome subassembly that is a key and integral unit in the function of the replisome during DNA replication. These proteins, isolated from diverse organisms, have been the subjects of structural studies as part of a broad effort to relate their structure to their function.The replicative helicases show common hexameric architecture. As revealed by electron micrographs, the DnaB helicase from Escherichia coli exists as an equilibrium mixture of two structural shapes: a hexamer with sixfold symmetry and a trimer of dimers with threefold symmetry (1). The hexamers are stabilized by Mg 2ϩ ; the equilibrium between the two ring forms can be shifted by the addition of nucleotides, such as ATP␥S, which favors a threefold symmetric ring (2).Key crystal structures of the active helicase domain of the phage T7 gene 4 protein revealed a hexameric structure whose subunit arrangements showed some deviation from a sixfold symmetry. The variation was ascribed to structural disruption postulated to accommodate stepwise ATP binding and hydrolysis, now reflected in this ''trapped'' structure (3). Threedimensional reconstruction and electron microscopy images of the intact T7 gp4 helicase͞primase also revealed a hexameric structure with sixfold symmetry (4). Nuclease protection and cross-linking experiments suggest that ssDNA passes through the hole in the center of the hexameric ring. Electron microscopy examination of the SV40 large T antigen (5) identified the helicase domains also as lying within a hexameric ring in an orientation identical to that seen by means of electron microscopic reconstruction of the bacteriophage T7 gp4 helicase (6).The crystal structure of the intact bifunctional helicase͞ primase from phage T7, however, revealed a heptameric ring (7). Retrospective electron microscopy showed that both hexameric and heptameric oligomeric forms were present. The heptameric ring, which contains the helicase domain, is relatively flat, but the adjoining subunits do not show true ...

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Section: Resultssupporting

confidence: 70%

Architecture of the bacteriophage T4 primosome: Electron microscopy studies of helicase (gp41) and primase (gp61)

Norcum

Warrington

Spiering

et al. 2005

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

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“…This hexameric form of rho is thought to represent the physiologically relevant association state and appears to be the form that binds to long RNA chains in solution Gogol et al, 1991). Also, the rho hexamer, when bound to long RNA, displays the ATPase activity that is required for rhodependent transcription termination (Richardson & Conaway, 1980;McSwiggen et al, 1988).…”

Section: Resultsmentioning

confidence: 99%

“…The dodecamer appears to consist of two planar hexagons stacked on top of each other (see also Gogol et al, 1991).…”

Section: Rho Complexed With Short Rna Ligandsmentioning

confidence: 99%

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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“…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).…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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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Structure and assembly of the Escherichia coli transcription termination factor rho and its interactions with RNA I. Cryoelectron microscopic studies

Cited by 97 publications

References 21 publications

Architecture of the bacteriophage T4 primosome: Electron microscopy studies of helicase (gp41) and primase (gp61)

Architecture of the bacteriophage T4 primosome: Electron microscopy studies of helicase (gp41) and primase (gp61)

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

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

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