Protein Science, Vol 1, Issue 7 850-860, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

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

J. GEISELMANN and P. H. VON-HIPPEL
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403

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(a) {complex} 3 x 10(6) M(-1)) and three weak (K(a) {complex} 10(5) M(-1)) binding sites for ATP. The binding constant of the weak binding sites is just the reciprocal of the enzymatic K(m) 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(3) to a pseudo-D(3) 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. 1, 861-873).
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