Mutational analysis and secondary structure model of the RNP1-like sequence motif of transcription termination factor Rho.
The function of transcription termination factor Rho from Escherichia coli is dependent upon its ability to bind to specific sites on nascent RNA molecules. The roles of 19 individual amino acid residues (Ile49 to Ser67) in and near a phylogenetically conserved sequence segment of Rho that is similar to the RNP1 motif found in many RNA-binding proteins were examined by testing the phenotypic consequences of mutational changes that were introduced into rho by a random-sequence cassette mutagenesis procedure. The tests of each mutant included the ability of the cells to survive at 42 degrees C in the absence of wild-type rho, the efficiency of termination at a Rho-dependent terminator (lambdatR1) in vivo, the relative level of expression of the mutant protein, and the ability of some of the mutant proteins to bind RNA. The results revealed that residues in the RNP1-like sequence of DGFGFLR (residues 60 to 66) were more important than residues 49 to 59 for termination function and RNA binding, and identified three residues that were particularly sensitive to mutation: Asp60, Phe62 and Arg66. The properties of the mutants are consistent with a secondary structure model, derived from phylogenetic analysis, that has the RNP1-like sequence on one of the three beta-strands of an antiparallel beta-sheet with Asp60 and Gly61 in a turn and the side-chains of Phe62, Phe64 and Arg66 accessible on the same face of the beta-structure for interaction with RNA.
Martinez
A
A
Department of Chemistry, Indiana University, Bloomington, 47405, USA.
Opperman
T
T
Richardson
J P
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000737:chemistry*; D017930:Genes, Reporter; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012234:Rho Factor / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013696:Temperature; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D016601:RNA-Binding Proteins; C082384:RNP1 protein, S cerevisiae; D012234:Rho Factor; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA