ATP-induced changes in the binding of RNA synthesis termination protein rho to RNA

Galluppi, Gerald R.; Richardson, John P.

doi:10.1016/s0022-2836(80)80016-7

Cited by 132 publications

(102 citation statements)

References 16 publications

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“…4, B and C). Thus, these results with natural transcripts as well as those with synthetic polymers showed that extensive protection of loop regions in the RNA-BD correlates with high affinity binding of these polymers to Rho (17,27).…”

Section: Cleavage Of Rho By Hydrogen Peroxide Andmentioning

confidence: 66%

“…We first used the C-terminal-specific antiserum to detect the fragments arising from cleavage in the RNA-BD. Rho is known to bind to single-stranded C-rich RNA and DNA molecules (17,21). A major part of this interaction is believed to occur in an extensive cleft formed by an arrangement of the RNA-BDs in hexameric Rho.…”

Section: Cleavage Of Rho By Hydrogen Peroxide Andmentioning

confidence: 99%

“…This model places the six RNA-BDs at one end of a ring-shaped structure with a C 6 symmetry. Although the precise orientation of the RNA binding clefts of the individual domain is uncertain, they are known to come together in the hexamer to form a single continuous cleft that is large enough to protect 60 nucleotides of poly(C) from cleavage by RNase A (4,17).…”

mentioning

confidence: 99%

“…However, no direct demonstration has been made yet to identify a distinct RNA-specific site on Rho. The binding of poly(dC) by hexameric Rho is competitive with the binding of poly(C) (17), and oligo(dC) binds to the isolated RBA-BD almost as well as does oligo(C) (21). Thus the proposed primary site is very likely composed of the RNA-BD from multiple subunits.…”

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confidence: 99%

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Identification of an RNA-binding Site in the ATP Binding Domain of Escherichia coli Rho by H2O2/Fe-EDTA Cleavage Protection Studies

Wei

Richardson

2001

Journal of Biological Chemistry

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Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA binding domain of known structure as well as an ATP binding domain for which a structure has been proposed based on homology modeling. Treatment of Rho with H 2 O 2 in the presence of Fe-EDTA caused single-cut cleavage at a number of points that coincide with solvent-exposed loops in both the known and predicted structures, thereby providing support for the validity of the tertiary and quaternary structural models of Rho. The binding of ATP caused one distinct change in the cleavage pattern, a strong protection at a cleavage point in the P-loop of the ATP binding domain. Binding of RNA and single-stranded DNA (poly(dC)) caused strong protection at several accessible parts of the oligosaccharide/oligonucleotide binding (OB) fold in the RNA binding domain. RNA molecules but not DNA molecules also caused a strong, ATP-dependent protection at a cleavage site in the predicted Q-loop of the ATP binding domain. These results suggest that Rho has two distinct binding sites for RNA. Besides the site composed of multiples of the RNA binding domain, to which singlestranded DNA as well as RNA can bind, it has a separate, RNA-specific site on the Q-loop in the ATP binding domain. In the proposed quaternary structure of Rho, the Q-loops from the six subunits form the upper entrance to the hole in the ring-shaped hexamer through which the nascent transcript is translocated by actions coupled to ATP hydrolyses.Transcript termination factor Rho is essential for orderly gene expression in many bacteria (1, 2). In Escherichia coli, Rho functions as a homohexamer of a 419-amino acid polypeptide (3-6). Both sequence and functional analyses have shown that Rho polypeptide has two distinct functional domains; one domain is the N-terminal RNA binding domain of residues 1-130 (RNA-BD), 1 and the other is C-terminal adenosine triphosphate binding domain of residues 131-419 (ATP-BD). Sequence comparison of Rho with other proteins indicates that it has ribonucleoprotein-like motifs in the RNA-BD and several motifs that are characteristic of ATPases in the ATP-BD (reviewed in Ref. 7). High resolution structures of the RNA-BD by itself (8, 9) and in a complex with oligo(C 9 ) (10) reveal that it contains a classic OB-fold motif that can form a stable complex with RNA (11,12). A model for the tertiary structure of the Rho ATP-BD has also been proposed based on the close sequence similarity of that part of Rho with the corresponding part of the ␣ and ␤ subunits of the F 1 -ATPase (13).A model for the quaternary structure of hexameric Rho has also been proposed (7, 13, 14) based on its sequence similarity and morphological resemblance to the F 1 -ATPase (5, 6, 15). More recently, a low resolution, three-dimensional model of Rho has been constructed from the analysis of electron microscopic images of Rho stained with uranyl acetate (16). This...

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Section: Cleavage Of Rho By Hydrogen Peroxide Andmentioning

confidence: 66%

Section: Cleavage Of Rho By Hydrogen Peroxide Andmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of an RNA-binding Site in the ATP Binding Domain of Escherichia coli Rho by H2O2/Fe-EDTA Cleavage Protection Studies

Wei

Richardson

2001

Journal of Biological Chemistry

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“…Rho carries an RNA-dependent ATPase activity that displays an RNA cofactor requirement similar to that of rho-dependent termination: i.e., a rho binding site on the RNA that is relatively devoid of stable secondary structure and relatively rich in cytosine residues. 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.…”

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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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Pausing of RNA polymerase molecules during in vivo transcription of the SV40 leader region.

Skolnik-David

Aloni

1983

The EMBO Journal

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Viral transcription complexes were isolated from SV40‐infected cells and incubated in vitro in the presence of [alpha‐32P]UTP to allow elongation of the promoter‐proximal RNA up to the attenuation sites. The 94 nucleotide attenuated RNA (spanning nucleotides 243‐336) was purified, digested with RNase T1 and fingerprinted. The labeled oligonucleotides were then isolated, digested with RNase T2 and their base composition was determined. Based on these analyses 10 consecutive oligonucleotides, spanning residues 259‐336, were identified. As the in vivo synthesized oligonucleotides are unlabeled the junctions between labeled and unlabeled oligonucleotides define the in vivo pause sites of RNA polymerase molecules. The characterization of the 10 radioactive spots and their relative intensities allowed the localization of two in vivo pause sites: one at 13‐16 nucleotides downstream from the major initiation site presumably at the initial opening of the DNA helix and the second at approximately 40 nucleotides downstream from the major initiation site, just past a GC‐rich region of dyad symmetry. It is postulated that pausing of RNA polymerase molecules in the leader region is an essential process in the control of SV40 late transcription.

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ATP-induced changes in the binding of RNA synthesis termination protein rho to RNA

Cited by 132 publications

References 16 publications

Identification of an RNA-binding Site in the ATP Binding Domain of Escherichia coli Rho by H2O2/Fe-EDTA Cleavage Protection Studies

Identification of an RNA-binding Site in the ATP Binding Domain of Escherichia coli Rho by H2O2/Fe-EDTA Cleavage Protection Studies

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

Pausing of RNA polymerase molecules during in vivo transcription of the SV40 leader region.

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