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

Abstract

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...

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

“…In this paper and its companion (Geiselmann & von Hippel, 1992) we have shown that the rho hexamer displays functional asymmetry with respect to the binding of both substrate (ATP) and cofactor (RNA). In particular, three of the six protomers of the rho hexamer (or 6 out of 12 promoters of the rho dodecamer) bind ATP and RNA much more tightly than do the remaining sites.…”

“…We calculate a 95% confidence interval of the fit parameter, taking into account the covariance of the fit parameter with the other estimated parameters as described in Geiselmann and von Hippel (1992). Binding constants of 50 x lo6 M" or greater cannot be reliably estimated from titration experiments carried out at the rho concentrations used here.…”

“…We have used these results to characterize some aspects of the binding interactions of rho with RNA, and, in particular, have observed changes in the state of association and the conformation of rho that result from RNA binding. The combination of these data with experiments on ATP binding (Geiselmann & von Hippel, 1992) and with published properties of the rho-RNA system allows us to propose a physical model for how rho might catalyze transcript termination (Geiselmann et al, in prep. ).…”

A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.

“…It has been shown that Rho exists under physiological conditions as a hexamer of identical subunits (19-23), organized as a trimer of asymmetric dimers with overall D3 symmetry (24). This symmetry is reflected functionally in the presence of three strong and three weak ATP (substrate) and three strong and three weak RNA (cofactor) binding sites per Rho hexamer (25)(26)(27)(28) (37,38), and subunit interaction sites have been proposed in the C-terminal region (35). Rho monomers associate to form a hexamer with D3 symmetry under physiological conditions.…”

“…It has been shown that Rho exists under physiological conditions as a hexamer of identical subunits (19)(20)(21)(22)(23), organized as a trimer of asymmetric dimers with overall D3 symmetry (24). This symmetry is reflected functionally in the presence of three strong and three weak ATP (substrate) and three strong and three weak RNA (cofactor) binding sites per Rho hexamer (25)(26)(27)(28) (37,38), and subunit interaction sites have been proposed in the C-terminal region (35 Six molecules of ATP can bind to the Rho hexamer; three bind with high affinity (25) and three bind 20-to 30-fold more weakly (26). Thus ATP binding creates (or induces) an asymmetry within the Rho hexamer and the smallest repeating unit of Rho can be considered to be a dimer of nonequivalent subunits in which one of the monomers allows tight binding of ATP and the other is in a low-ATP-affinity state.…”