A 2'-methyl or 2'-methylene group at G+1 in precursor tRNA interferes with Mg2+ binding at the enzyme-substrate interface in E-S complexes of E. coli RNase P.
We analyzed processing of precursor tRNAs carrying a single 2'-deoxy, 2'-OCH(3), or locked nucleic acid (LNA) modification at G+1 by Escherichia coli RNase P RNA in the absence and presence of its protein cofactor. The extra methyl or methylene group caused a substrate binding defect, which was rescued at higher divalent metal ion (M(2+)) concentrations (more efficiently with Mn(2+) than Mg(2+)), and had a minor effect on cleavage chemistry at saturating M(2+) concentrations. The 2'-OCH(3) and LNA modification at G+1 resulted in higher metal ion cooperativity for substrate binding to RNase P RNA without affecting cleavage site selection. This indicates disruption of an M(2+) binding site in enzyme-substrate complexes, which is compensated for by occupation of alternative M(2+) binding sites of lower affinity. The 2'-deoxy modification at G+1 caused at most a two-fold decrease in the cleavage rate; this mild defect relative to 2'-OCH(3) and LNA at G+1 indicates that the defect caused by the latter two is steric in nature. We propose that the 2'-hydroxyl at G+1 in the substrate is in the immediate vicinity of the M(2+) cluster at the phosphates of A67 to U69 in helix P4 of E. coli RNase P RNA.
Cuzic
Simona
S
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, Germany.
Hartmann
Roland K
RK
D000225:Adenine / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002413:Cations, Divalent / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006147:Guanine / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008697:Methane / Q000737:chemistry / Q000378:metabolism*; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012322:RNA Precursors / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics*; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D012322:RNA Precursors; D006147:Guanine; D012343:RNA, Transfer; D043262:Ribonuclease P; D008274:Magnesium; D000225:Adenine; D008697:Methane