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 nascen t RNA molecules. The roles of 19 individual amino acid residues (IIe49 to Ser67) in and near a phylogenetically conserved sequence segment o f Rho that is similar to the RNP1 motif found in many RNA-binding prot eins were examined by testing the phenotypic consequences of mutationa l changes that were introduced into who by a random-sequence cassette mutagenesis procedure. The tests of each mutant included the ability o f the cells to survive at 42 degrees C in the absence of wild-type rho , the efficiency of termination at a Rho-dependent terminator (lambda tR1) in vivo, the relative level of expression of the mutant protein, and the ability of some of the mutant proteins to bind RNA. The result s revealed that residues in the RNP1-like sequence of DGFGFLR (residue s 60 to 66) were more important than residues 49 to 59 for termination function and RNA binding, and identified three residues that were par ticularly sensitive to mutation: Asp60, Phe62 and Arg66. The propertie s of the mutants are consistent with a secondary structure model, deri ved from phylogenetic analysis, that has the RNP1-like sequence on one of the three beta-strands of an antiparallel beta-sheet with Asp60 an d Gly61 in a turn and the side-chains of Phe62, Phe64 and Arg66 access ible on the same face of the beta-structure for interaction with RNA. (C) 1996 Academic Press Limited