RP-PHOSPHOROTHIOATE MODIFICATIONS IN RNASE-P RNA THAT INTERFERE WITH TRANSFER-RNA BINDING

We have used Rp-phosphorothioate modifications and a binding interfere nce assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA, Total (100%) Rp-pho sphorothioate modification at A, C or G positions of RNase P RNA stron gly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions, Partially modified E.coli RNase P RNAs were separated into tRNA binding and nonbinding fractions by gel reta rdation. Rp-phosphorothioate modifications that interfered with tRNA b inding were found 5' of nucleotides A(67), G(68), U-69, C-70, C-71, G( 72), A(130), A(132), A(248), A(249), G(300), A(317), A(330), A(352), C -353 and C-354. Manganese rescue at positions U-69, C-70, A(130) and A (132) identified, for the first time, sites of direct metal ion coordi nation in RNase P RNA, Most sites of interference are at strongly cons erved nucleotides and nine reside within a long-range base-pairing int eraction present in all known RNase P RNAs, In contrast to RNase P RNA , 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E.coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.