Residues in the RNP1-like sequence motif of Rho protein are involved in RNA-binding affinity and discrimination.
The termination of transcription in Escherichia coli by action of Rho factor is dependent on the ability of this homohexameric protein to make productive interactions with the nascent RNA molecule to be terminated. The roles of two residues in a phylogenetically conserved sequence motif in the RNA-binding domain of Rho, Asp60 and Phe62, were analyzed by studies of the biochemical properties of pure mutant proteins. F62S Rho had greatly reduced affinity for lambda cro RNA, very poor ability to terminate transcription in vitro by itself and only partial termination activity (at a level consistent with its in vivo defect) in the presence of NusG. D60G Rho had a high affinity for lambda cro RNA but a much lower ability to discriminate against RNA molecules lacking cis-acting Rho-utilization sequences, and a reduced efficiency of termination that was not improved by NusG. These results indicate a major role for Phe62 in stabilizing the binding of Rho to RNA through hydrophobic interactions, while Asp60 provides an electrostatic repulsive force that allows a rapid dissociation of non-productive complexes with RNA.
Martinez
A
A
Department of Chemistry, Indiana University, Bloomington, 47405, USA.
Burns
C M
CM
Richardson
J P
JP
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D017124:Conserved Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012234:Rho Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic
D017931:DNA Primers; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C082384:RNP1 protein, S cerevisiae; D012234:Rho Factor; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D010649:Phenylalanine; D012313:RNA; D000251:Adenosine Triphosphatases