STRUCTURAL AND FUNCTIONAL DISSECTIONS OF TRANSCRIPTION TERMINATION FACTOR-RHO BY RANDOM MUTAGENESIS

Transcription termination factor rho from Escherichia coli is a homohe xamer of 419 amino acid subunits and catalyzes an ATP-dependent releas e of nascent RNA transcripts. A rho monomer has three distinct domains functioning independently at the first approximation: the amino-termi nal one quarter containing a primary RNA-binding site, the central 270 -amino acids region constituting an ATP-binding domain with homologies to F-1-ATPase, and the carboxy-terminal remainder with unknown functi on(s). To further delineate the structural and functional organization s of rho protein, we undertook its random mutagenesis using error-pron e polymerase chain reactions with the carboxy-terminal 100-amino acid region chosen as the initial target. From 14 mutants identified, rho p rotein was purified and characterized in vitro. Of these, 11 mutants a re defective in termination in vivo and show decreased activities in v arious partial functions examined: ATP binding; RNA binding; and ATPas e activities dependent on three cofactors with decreasing efficacies, poly(C), hero RNA and poly(U). A few of them are also affected in the putative secondary RNA-binding site that is functionally coupled to AT P hydrolysis. By contrast, the three other mutants are hyperactive in termination, poly(U)-dependent ATPase activity, and RNA interaction at the primary site. Ln these properties, the hyper-terminating mutants strikingly resemble the ''super rho'' mutant formerly found in the ami no-terminal domain. Taken together, these findings indicate that the c arboxy-terminal region plays a pivotal role in functionally coupling t he RNA and ATP-binding domains, plausibly by acting as an interface fo r their interaction within or across individual subunits. In light of the reported X-ray crystallographic structure of R-ATPase, we propose a model for the tertiary and quaternary structure of rho that is consi stent with the observed mutational effects as well as a number of stru ctural and functional properties characteristic of rho. (C) 1995 Acade mic Press Limited