Transcription termination factor rho contains three noncatalytic nucleotide binding sites

The active form of transcription termination factor rho from Escherichia co li is a homohexamer, but several studies suggest that the six subunits of t he hexamer are not functionally identical. Rho has three tight and three we ak ATP binding sites. Based on our findings, we propose that the tight nucl eotide binding sites are noncatalytic and the weak sites are catalytic. In the presence of RNA, the rho-catalyzed ATPase rate is fast, close to 30 s(- 1). However, under these conditions the three tightly bound nucleotides dis sociate from the rho hexamer at a slow rate of 0.02 s(-1), indicating that the three tight nucleotide binding sites of rho do not participate in the f ast ATPase turnover. These slowly exchanging nucleotide binding sites of rh o are capable of hydrolyzing ATP, but the resulting products (ADP and pi) b ind tightly and dissociate from rho about 1500 times slower than the fast A TPase turnover. Both RNA and excess ATP in solution are necessary for stabi lizing nucleotide binding at these sites. In the absence of RNA or when sol ution ATP is hydrolyzed to ADP, a faster dissociation of nucleotides was ob served. Based on these results, we pro pose that the rho hexamer is similar to the F-1-ATPase and T7 DNA helicase-containing noncatalytic sites that d o not participate in the fast ATPase turnover. We propose that the three ti ght sites on rho are the noncatalytic sites and the three weak sites are th e catalytic sites.