RHO-DEPENDENT TERMINATION AND CONCOMITANT NTPASE ACTIVITY REQUIRES A SPECIFIC, INTACT RNA REGION

The specific DNA and RNA requirements for rho-dependent transcription termination were investigated in vitro. As a model, templates containing the rho-dependent terminator of the Escherichia coli trp operon, trp t'' were used. Templates containing the trp t'' region direct specific rho-dependent termination in vitro, with concomitant stimulation of the rho NTPase [nucleoside triphosphatase] activity, and deletion of the trp t'' region results in templates that do not induce rho-dependent termination or rho NTPase activity. Addition of RNase T1 to transcription reactions specifically eliminated transcription termination and rho NTPase activity. These results demonstrate the requirement for a specific RNA component within the trp t'' transcript necessary for NTPase activation and rho-dependent transcription termination. Active transcription was not a prerequisite for rho NTPase activation; trp t'' RNA (rho-terminated transcripts) and read-through transcripts, which contain the trp t'' region, activated the rho NTPase when rho was added after inhibition of transcription. As is true for synthetic polynucleotides known to activate the rho NTPase, the trp t'' region has few G resides. This reduces the potential for the formation of stable secondary structures in the RNA transcript, and may be a determinant of sites specifying rho-dependent termination of transcription. The implications of this are discussed in light of the lack of significant sequence homologies between rho-dependent transcription termination sites.