Verification of phylogenetic predictions in vivo and the importance of thetetraloop motif in a catalytic RNA

M1 RNA, the catalytic subunit of Escherichia coil RNase P, forms a secondar y structure that includes five sequence variants of the tetraloop motif, Si te-directed mutagenesis of the five tetraloops of M1 RNA, and subsequent st eady-state kinetic analysis in vitro, with different substrates in the pres ence and absence of the protein cofactor, reveal that (i) certain mutants e xhibit defects that vary in a substrate-dependent manner, and that (ii) the protein cofactor can correct the mutant phenotypes in vitro, a phenomenon that is also substrate dependent. Thermal denaturation curves of tetraloop mutants that exhibit kinetic defects differ from those of wild-type M1 RNA, Although the data collected in vitro underscore the importance of the tetr aloop motif to M1 RNA function and structure, three of the five tetraloops we examined in vivo are essential for the function off. coil RNase P, The k inetic data in vitro are not in total agreement with previous phylogenetic predictions but the data in vivo are, as only mutants in those tetraloops p roposed to be involved in tertiary interactions fail to complement in vivo. Therefore, the tetraloop motif is critical for the stabilization of the st ructure of M1 RNA and essential to RNase P function in the cell.