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

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Abstract
M1 RNA, the catalytic subunit of Escherichia coli RNase P, forms a secondary structure that includes five sequence variants of the tetraloop motif. Site-directed mutagenesis of the five tetraloops of M1 RNA, and subsequent steady-state kinetic analysis in vitro, with different substrates in the presence and absence of the protein cofactor, reveal that (i) certain mutants exhibit 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 tetraloop motif to M1 RNA function and structure, three of the five tetraloops we examined in vivo are essential for the function of E. coli RNase P. The kinetic data in vitro are not in total agreement with previous phylogenetic predictions but the data in vivo are, as only mutants in those tetraloops proposed to be involved in tertiary interactions fail to complement in vivo. Therefore, the tetraloop motif is critical for the stabilization of the structure of M1 RNA and essential to RNase P function in the cell.