Nucleic Acids Research Advance Access published online on September 30, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp775
RNA |
Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany
*To whom correspondence should be addressed. Tel: +49 6421 2825827; Fax +49 6421 2825854; Email: roland.hartmann{at}staff.uni-marburg.de
Received July 13, 2009. Revised August 31, 2009. Accepted September 2, 2009.
We analyzed cleavage of precursor tRNAs with an LNA, 2'-OCH3, 2'-H or 2'-F modification at the canonical (c0) 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 c0 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'-OCH3 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 c0 and m+1 sites had different pH profiles, with a higher Mg2+ requirement for c0 versus m+1 cleavage. The strong catalytic defect for LNA and 2'-OCH3 supports a model where the extra methylene (LNA) or methyl group (2'-OCH3) causes a steric interference with a nearby bound catalytic Mg2+ 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.
Present address: Dr Simona Cuzic-Feltens, Martin-Luther Universität Halle, Institut für Biochemie und Biotechnologie, Kurt-Mothes Strasse, 306120 Halle (Saale), Germany.