| 19793868 |
Cuzic-Feltens S, Weber MH, Hartmann RK: Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester. Nucleic Acids Res. 2009 Dec;37(22):7638-53. Epub .
We analyzed cleavage of precursor tRNAs with an LNA, 2'-OCH (3), 2'-H or 2'-F modification at the canonical (c (0)) site by bacterial RNase P. We infer that the major function of the 2'-substituent at nt -1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c (0) site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3' -endo ribose and without the H-bond donor function of the 2'-substituent. LNA and 2'-OCH (3) suppressed processing at the major aberrant m (-)(1) site; instead, the m (+1) (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c (0) and m (+1) sites had different pH profiles, with a higher Mg (2+) requirement for c (0) versus m (+1) cleavage. The strong catalytic defect for LNA and 2'-OCH (3) supports a model where the extra methylene (LNA) or methyl group (2'-OCH (3)) causes a steric interference with a nearby bound catalytic Mg (2+) during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects. |
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