Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer

Geiselmann, Johannes; Yager, Thomas D.; Gill, Stanley C.; Calmettes, P.; Hippel, Peter H. von

doi:10.1021/bi00116a017

Cited by 77 publications

(90 citation statements)

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“…We therefore conclude that rho forms a hexamer in the presence of (rC)70 and that (rC)70 binds to this hexamer with a 1 : 1 stoichiometry. This result confirms earlier determinations of the association state of rho and of the RNA site size within the rho-RNA complex and suggests that the rho hexamer is the physiologically relevant species at the protein concentrations that prevail in vivo (see Geiselmann et al, 1992b). (We note also a secondary effect, which appears to be a weak association of rho hexamers that have RNA bound to them.…”

Section: Rna Binding Measured In the Analytical Ultracentrifugesupporting

confidence: 90%

“…Combination of the S and D values of a homogeneous species allows calculation of its molecular weight via the Svedberg equation and confirms that rho forms a dodecamer under these conditions (Geiselmann et al, 1992b). The dodecameric form of rho is generated by binding oligo(rC) (or oligo(dC)) ligands that are 9-20 nucleotide residues in length (see also Fig.…”

Section: Rho Complexed With Short Rna Ligandsmentioning

confidence: 62%

“…The physiological association state of rho Rho binds (rC),o as a hexamer We have previously described conditions under which a stable, homogeneous hexamer of rho exists in solution (Geiselmann et al, 1992b). 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).…”

Section: Resultsmentioning

confidence: 99%

“…In Figure 2 we plot the integral distribution of the sedimentation coefficient for a number of different rho-RNA complexes (see van Holde & Weischet [ 19781 for details of this type of analysis of a sedimentation boundary). The data for rho alone (which exists as a hexamer under these conditions [see Geiselmann et al, 1992b]), and for rho bound to (rC)70 at a stoichiometry of one ligand per rho hexamer, are shown in the top panel of Figure 2. The rho-RNA complex sediments faster than the unliganded rho hexamer, but considerably more slowly than does the rho dodecamer described below.…”

Section: Resultsmentioning

confidence: 99%

“…2. ) Numerous experiments have shown that short RNA ligands drive rho from the hexamer to a dodecamer association state (see above and Geiselmann et al, 1992b). Sedimentation velocity experiments, carried out at different rho:RNA ratios, demonstrate that the strong RNA binding sites on the dodecamer saturate at binding stoichiometries of about six RNA ligands per dodecamer.…”

Section: Rna Binding Measured In the Analytical Ultracentrifugementioning

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: Rna Binding Measured In the Analytical Ultracentrifugesupporting

confidence: 90%

Section: Rho Complexed With Short Rna Ligandsmentioning

confidence: 62%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Rna Binding Measured In the Analytical Ultracentrifugementioning

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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Analytical Ultracentrifugation of Model Nanoparticles: Comparison of Different Analysis Methods

Mittal

Völkel

Cölfen

2010

Macromolecular Bioscience

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Sedimentation analysis of nanoparticle (ZrO(2) and SiO(2)) suspensions of different particle sizes in various solvents as nanoparticle model systems was carried out using interference optics in the analytical ultracentrifuge. The particles differed in their morphology: SiO(2) particles were spherical, whereas ZrO(2) particles were in one case spherical and in the second case non-spherical and spherical. Different analysis programs, based on different principles of data analysis, were used for the evaluation of the size distributions of these particles, viz. SEDFIT [ls-g*(s), c(s)], UltraScan (vHW, 2DSA-MC), SedAnal (dcdt) and VelXLAI (GFL) method and SedAnal, in order to ascertain the benefits and limitations of these analysis methods in characterising the examined nanoparticles.

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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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Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer

Cited by 77 publications

References 41 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

Analytical Ultracentrifugation of Model Nanoparticles: Comparison of Different Analysis Methods

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

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