Escherichia coli transcription termination factor rho. I. ATPase activation by oligonucleotide cofactors

Rho protein is required to bring about RNA release from Escherichia coli transcription complexes paused at specific (rho-dependent) termination sites. Rho functions in termination as a hexamer of identical subunits arranged in D3 symmetry, with each rho subunit carrying an RNA- and an ATP-binding site. The detailed mechanism of rho-catalyzed transcript release remains to be determined, but it is clear that the RNA-dependent ATPase activity that is stimulated by interaction with the nascent transcript is essential to the termination function of rho. In this study, we have used short (8-10 nucleotide residues) synthetic ribo-oligonucleotides to model the interaction of segments of the RNA cofactor with rho. A poly(dC) enhancement procedure was used to permit the measurement of steady state ATPase parameters. We show that (i) ATPase activation is cofactor composition- and sequence-dependent; (ii) at least 60% of the residues of these short RNA cofactors must be cytosine to produce maximal rho ATPase activation; (iii) oligo(rU,rC) cofactors with the rU residues located at the 5' termini of the oligomer are much better ATPase cofactors than oligomers containing rC residues only; (iv) this enhanced stimulation is not observed if the rU residues are replaced by rA residues; (v) this cofactor activity relative to oligo(rC) is reversed if the rU residues are placed at the 3' terminus of RNA oligomer; and (vi) these nucleotide sequence and composition effects do not appear to be functions of K+ or Mg2+ concentration. These ATPase activation results are correlated with the binding to rho of oligonucleotide cofactors in the accompanying paper (Wang, Y., and von Hippel, P. H. (1993) J. Biol. Chem. 268, 13947-13955).