Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester.
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.
Cuzic-Feltens
Simona
S
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
Weber
Michael H W
MH
Hartmann
Roland K
RK
D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D055162:Biocatalysis; D029968:Escherichia coli Proteins / Q000378:metabolism*; D020035:Holoenzymes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism; D012353:RNA, Transfer, Gly / Q000737:chemistry* / Q000378:metabolism; D043262:Ribonuclease P / Q000378:metabolism*
D029968:Escherichia coli Proteins; D020035:Holoenzymes; D009841:Oligonucleotides; D010710:Phosphates; D012322:RNA Precursors; D012353:RNA, Transfer, Gly; C477371:locked nucleic acid; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium