Interactions of Escherichia coli transcription termination factor rho with RNA

McSwiggen, James A.; Bear, David G.; Hippel, Peter H. von

doi:10.1016/0022-2836(88)90305-1

Cited by 100 publications

(96 citation statements)

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“…This may reflect some concentration-dependent aggregation due to the formation of bridged complexes in which single RNA molecules are bound to different rho hexamers (as seen with rho-[rCId5 complexes described below). It could also reflect a general affinity of rho-RNA complexes for one another, which would be consistent with the observation that hexamers bind cooperatively to poly(rC) McSwiggen et al, 1988). We note that the hexameric limit is attained at the low concentration end of the sedimentation boundary in our experiments.…”

Section: Resultssupporting

confidence: 90%

“…When the large ligand binding equation is used we obtain a good fit of the data and estimate a binding constant of lo7 M" (per mole nucleotide residue) and a cooperativity parameter of -30. The resulting value of the logarithm of the Kw product (8.5) is consistent with the results of McSwiggen et al (1988), who found log(Kw) to fall between 8.3 and 9.5.…”

Section: Rna Binding Monitored By CD Spectroscopysupporting

confidence: 89%

“…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 binding site size of rho for RNA polymers, as determined by McSwiggen et al (1988) using fluorescently labeled RNA polymers and in this work using CD titration with poly(rC), is 11-12 nucleotide residues per rho protomer. (We note that the results of McSwiggen et al cited here have been corrected to take into account the more accurate extinction coefficient of rho measured by Geiselmann et al [ 1992bI .)…”

Section: Binding Stoichiometriesmentioning

confidence: 88%

“…In order to extract estimates of the binding constant, K , and the cooperativity parameter, w , these data should instead be fit to the large ligand binding equation that takes overlapping binding sites into account (McGhee & von Hippel, 1974;McSwiggen et al, 1988). When the large ligand binding equation is used we obtain a good fit of the data and estimate a binding constant of lo7 M" (per mole nucleotide residue) and a cooperativity parameter of -30.…”

Section: Rna Binding Monitored By CD Spectroscopymentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 90%

Section: Rna Binding Monitored By CD Spectroscopysupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Binding Stoichiometriesmentioning

confidence: 88%

Section: Rna Binding Monitored By CD Spectroscopymentioning

confidence: 99%

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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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Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. I. Binding of ATP

Geiselmann

Hippel

1992

Protein Science

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Escherichia coli transcription termination factor rho is an RNA-dependent ATPase, and ATPase activity is required for all its functions. We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer. This stoichiometry has been verified by a number of different techniques, including ultracentrifugation, ultrafiltration, and fluorescence titration studies. We have also shown that ATP can bind to isolated monomers of rho when the hexamer is dissociated with the mild denaturant myristyltrimethylammonium bromide, demonstrating that each protomer of rho carries an ATP binding site. The six binding sites that we observe in the rho hexamer are not equivalent; the hexamer contains three strong (K,, = 3 x lo6 M") and three weak (K,, = IO' M-I) binding sites for ATP. The binding constant of the weak binding sites is just the reciprocal of the enzymatic K , for ATP as a substrate; thus these weak sites, as well as the strong sites, can, in principle, take part in the catalytic cycle. The asymmetry induced (or manifested) by ATP binding reduces the symmetry of the rho hexamer from a D, to a pseudo-D, state. This "breakage" of symmetry has implications for the molecular mechanism of rho, because an asymmetric structure can lead to directional helicase activity by invoking directionally distinct RNA binding and release reactions (see Geiselmann, J., Yager, T.D., & von Hippel, P.H., 1992c, Protein Sci. I, 861-873).Keywords: ATP binding; Escherichia coli; rho; stoichiometry; symmetry; termination; transcription Rho protein of Escherichia coli is required for termination of transcription by RNA polymerase and for the release of the nascent transcript at specific rho-dependent termination sites. The 47 kD protomers of rho associate to a hexamer with D3 symmetry to form the functionally active and physiologically relevant form of this protein (Finger & Richardson, 1982; Geiselmann et al., 1992a,b). Rho (in the absence of RNA polymerase) can operate as a 5' --r 3' RNA-DNA helicase on appropriate substrates (Brennan et al., 1987(Brennan et al., , 1990. All the functions of rho involve the hydrolysis of nucleoside triphosphates; ATP is assumed to be the natural substrate and rho is therefore considered an ATPase.The ATPase activity of rho is absolutely dependent on the presence of an RNA cofactor. The highest level of ATPase activity is observed when rho is bound to a homopolymer of cytosine. In order to develop a molecular model of rho function in transcription termination, it is important to understand its interactions with its ATP substrate and RNA cofactors. The RNA binding properties of rho have been investigated in a number of laboratories (Lowery & Richardson, 1977b;Galluppi & Richardson, 1980;von Hippel et al., 1987) and are further characterized in the companion paper (Geiselmann et al., 1992~).The interactions of rho with ATP have been examined at the functional (Lowery & Richardson...

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In VitroCharacterization of Transcription Termination Factor Rho fromEscherichia coli rho(nusD)Mutants

Washburn

Stitt

1996

Journal of Molecular Biology

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Interactions of Escherichia coli transcription termination factor rho with RNA

Cited by 100 publications

References 26 publications

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

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

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. I. Binding of ATP

In VitroCharacterization of Transcription Termination Factor Rho fromEscherichia coli rho(nusD)Mutants

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