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

Abstract

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.

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

“…The interaction of rho with its other cofactor, RNA, is addressed in the accompanying paper (Geiselmann et al, 1992~). Binding and hydrolysis of ATP is required for all functions of the rho protein.…”

“…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~).…”

“…We repeated the titration of rho with &-ATP under the conditions described above, but in the presence of a saturating concentration of (dC),,. The apparent binding constant for &-ATP is three-to fourfold weaker than that observed in the absence of the (dC),, ligand, suggesting some allosteric coupling between the ATP and the oligonucleotide binding sites of rho (see Geiselmann et al, 1992~). The reliability of the fit involved in determining stoichiometry is reduced, due to the lower apparent binding constant, but the best fit remains the same: i.e., one &-ATP is bound per rho protomer.…”

“…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, 1977a,b;Galluppi & Richardson, 1980;Stitt & Webb, 1986;Stitt, 1988) and the structural level (Engel 8z Richardson, 1984;Bear et al, 1985;Dombroski & Platt, 1988; Dombroski et al, 1988a,b). These studies have shown that rho possesses a distinct ATP binding domain on each protomer (Dombroski et al, 1988a,b), but that only three protomers per hexamer appear to bind a molecule of ATP 850 ATP binding to rho protein 85 1 (Dombroski et al, 1988b;Stitt, 1988).…”

“…In order to achieve a directional helicase activity (Brennan et al, 1987(Brennan et al, , 1990 using elementary RNA binding and release reactions one has to invoke an asymmetric structure. Binding of the RNA ligand further reduces the symmetry of the (now) pseudo-D, symmetrical hexamer (Geiselmann et al, 1992~). The resulting structure provides the basis for a proposed physical model for the function of rho protein in transcription termination (Geiselmann et al, in prep.…”