title: Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover.
abstract: Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.
authors.affiliation: Department of Biochemistry, University of Rochester, New York 14642.
authors.forename: C A
authors.initials: CA
authors.lastname: Brennan
authors.forename: T
authors.initials: T
authors.lastname: Platt
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D016384:Consensus Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D012329:RNA, Bacterial / Q000378:metabolism; D016601:RNA-Binding Proteins; D012234:Rho Factor / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012492:Salts; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
chemical_list: D002352:Carrier Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D012234:Rho Factor; D012261:Ribonucleoproteins; D012492:Salts; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D007930:Leucine; D000409:Alanine