title: Structural and functional dissections of transcription termination factor rho by random mutagenesis.
abstract: Transcription termination factor rho from Escherichia coli is a homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts. A rho monomer has three distinct domains functioning independently at the first approximation: the amino-terminal one quarter containing a primary RNA-binding site, the central 270-amino acids region constituting an ATP-binding domain with homologies to F1-ATPase, and the carboxy-terminal remainder with unknown function(s). To further delineate the structural and functional organizations of rho protein, we undertook its random mutagenesis using error-prone polymerase chain reactions with the carboxy-terminal 100-amino acid region chosen as the initial target. From 14 mutants identified, rho protein was purified and characterized in vitro. Of these, 11 mutants are defective in termination in vivo and show decreased activities in various partial functions examined: ATP binding; RNA binding; and ATPase activities dependent on three cofactors with decreasing efficacies, poly(C), lambda cro RNA and poly(U). A few of them are also affected in the putative secondary RNA-binding site that is functionally coupled to ATP hydrolysis. By contrast, the three other mutants are hyperactive in termination, poly(U)-dependent ATPase activity, and RNA interaction at the primary site. In these properties, the hyper-terminating mutants strikingly resemble the "super rho" mutant formerly found in the amino-terminal domain. Taken together, these findings indicate that the carboxy-terminal region plays a pivotal role in functionally coupling the RNA and ATP-binding domains, plausibly by acting as an interface for their interaction within or across individual subunits. In light of the reported X-ray crystallographic structure of F1-ATPase, we propose a model for the tertiary and quaternary structure of rho that is consistent with the observed mutational effects as well as a number of structural and functional properties characteristic of rho.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D012234:Rho Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D012234:Rho Factor; D012313:RNA; D000255:Adenosine Triphosphate