Suppression of loss-of-function mutations in Escherichia coli ribonuclease P RNA (M1 RNA) by a specific base-pair...

A plasmid encoding ribonuclease P RNA of Escherichia coli (M1 RNA) was mutagenized with hydroxylamine in vitro and defective rnpB genes were identified by screening in an in vivo suppression assay. Defective rnpB sequences were mutagenized with a second round of hydroxylamine to restore activity. We report here that conversion of the C32.G48 base-pair of RNase P RNA to either C.A or U.G restored activity to defective rnpB genes bearing a variety of spatially distinct primary mutations. Disruption of this base-pair in an otherwise wild-type rnpB sequence increased the growth rate of the indicator strain E. coli FS101, consistent with the opening of C32.G48 during in vivo assembly of or catalysis by RNase P.