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.
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
Simona
S
Cuzic-Feltens
Michael H W
MH
Weber
Roland K
RK
Hartmann
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