A MUTATION IN THE ATP BINDING DOMAIN OF RHO ALTERS ITS RNA-BINDING PROPERTIES AND UNCOUPLES ATP HYDROLYSIS FROM HELICASE ACTIVITY

The Escherichia coli mutant rho201 was originally isolated in a geneti c screen for defects in rho-dependent termination. Cloning and sequenc ing of this gene reveals a single phenylalanine to cysteine mutation a t residue 232 in the ATP binding domain of the protein, This mutation significantly alters its RNA binding properties so that it binds trp t ' RNA 100-fold weaker than the wild type protein, with a K-d of approx imately 1.3 nM. Rho201 binds nonspecific RNA only 3-4 fold less tightl y than it binds trp t', while the wild type differential for these sam e RNAs is 10-20-fold. Curiously, rho201 displays in creased secondary site RNA activation, with a K-m for ribo(C)(10) of 0.6 mu M, compared to the wild type value of 3-4 mu M. Although rho201 and the wild type protein hydrolyze ATP similarly with poly(C), or trp t' RNA, as cofact ors, rho201 has a higher ATPase activity when activated by nonspecific RNA, Physically, rho201 displays an abnormal conformation detectable by mild trypsin digestion. Despite effective ATP hydrolysis, the rho20 1 mutant is a poor RNA:DNA helicase and terminates inefficiently on tr p t', The single F232C mutation thus appears to uncouple the protein's ATPase activity from its helicase function, so rho can no longer harn ess available energy for use in subsequent reactions.