Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding.
Ribose 2'-hydroxyls make a key contribution to the enormous structural and functional potential of RNA molecules. Here, we report the identification of 2'-deoxy modifications in the catalytic RNA subunit of RNase P from Escherichia coli that interfere with tRNA binding. This was accomplished by modification interference employing pools of RNase P RNA that carried a low level of Rp-deoxy-phosphorothioate (Rp-deoxyNMPalpha(S) ) modifications randomly distributed over its 380 nt. A gel retardation assay allowed us to separate RNase P RNA pools into tRNA-binding and nonbinding fractions. Differences in the intensity of phosphorothioate-specific iodine hydrolysis patterns of the two RNA fractions revealed positions where the Rp-deoxyNMPalpha(S) modification interferes with tRNA binding. A comparison with interference patterns obtained for the Rp-NMPalpha(S) modification alone has identified some 20 positions in the backbone of E. coli RNase P RNA where the functional defect caused by the Rp-deoxyNMPalpha(S) double modification is attributable to the 2'-deoxy modification (or possibly the C5 methyl group in the case of U residues because we used deoxyTMPalpha(S) for partial substitution of UMP). Most of the corresponding 2'-OH functions were localized in regions that have been reported to crosslink to photoreactive tRNA derivatives, suggesting that these 2'-hydroxyls are located along the tRNA binding interface of E. coli RNase P RNA. Our results indicate that the modification interference approach applied here will be useful generally to identify structurally and functionally important 2'-hydroxyls in large RNAs and ribozymes.
Hardt
W D
WD
Institut für Biochemie, Freie Universität Berlin, Germany.
Erdmann
V A
VA
Hartmann
R K
RK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013768:Tetrahymena / Q000235:genetics; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012322:RNA Precursors; D016337:RNA, Catalytic; D013873:Thionucleotides; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli