Escherichia coli transcription termination factor rho. II. Binding of oligonucleotide cofactors.

The relative binding affinities for rho of the oligonucleotide rho ATPase cofactors studied in the accompanying paper (Wang, Y., and von Hippel, P. H. (1993) J. Biol. Chem. 268, 13940-13946) have been determined by gel mobility shift and ultrafiltration binding analyses. We find that each rho hexamer carries three strong and three weak RNA-binding sites that differ approximately 10-fold in their affinities for oligonucleotide cofactors. Furthermore, in contrast to the sequence dependence of ATPase activation, we find that the binding affinities of these oligonucleotide cofactors for rho depend only on their cytosine content. In addition, we show that changes in the positions of rU residues in the oligo(rU,rC) cofactors (which significantly modulate the ATPase activity of rho) have no effect on binding affinities and that the addition of ATP, ADP, or the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate also does not change the binding affinities of the oligonucleotide cofactors for rho. Considered in the context of the coupling of the rho ATPase and RNA binding and release cycles, these results suggest that rC residues are required for the formation of stable rho-RNA complexes, whereas rU residues at the 5' termini of cofactors bound to rho initiate or facilitate the release of the RNA from the individual cofactor site as a consequence of ATP hydrolysis. Thus, both the tightness of the binding of RNA segments to the individual RNA-binding sites of rho and the rate of release of these segments from these sites are critical in controlling the ATPase rate of rho and probably also in modulating the function of this protein in transcript termination.