Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA

We have identified by nucleotide analog interference mapping (NAIM) exocycl ic NH2 groups of guanosines in RNase P RNA from Escherichia coli that are i mportant for tRNA binding. The majority of affected guanosines represent ph ylogenetically conserved nucleotides. Several sites of interference could b e assigned to direct contacts with the tRNA moiety, whereas others were int erpreted as reflecting indirect effects on tRNA binding due to the disrupti on of tertiary contacts within the catalytic RNA. Our results support the i nvolvement of the 2-NH2 groups of G292/G293 in pairing with C-74 and C-75 O f tRNA CCA-termini, as well as formation of two consecutive base triples in volving C-75 and A(76) Of CCA-ends interacting with G292/A258 and G291/G259 , respectively. Moreover, we present first biochemical evidence for two ter tiary contacts (L18/P8 and L8/P4) within the catalytic RNA, whose formation has been postulated previously on the basis of phylogenetic comparative an alyses, The tRNA binding interference data obtained in this and our previou s studies are consistent with the formation of a consecutive nucleotide tri ple and quadruple between the tetraloop L18 and helix P8, Formation of the nucleotide triple (G316 and A94:U104 in wild-type E, coil RNase P RNA) is a lso supported by mutational analysis. For the mutant RNase P RNA carrying a G94:C104 double mutation, an additional G316-to-A mutation resulted in a r estoration of binding affinity for mature and precursor tRNA.