| 17570824 |
Cuzic S, Hartmann RK: 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. Biol Chem. 2007 Jul;388(7):717-26.
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. |
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