Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.
AUTOR(ES)
Mobley, E M
RESUMO
Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=148707Documentos Relacionados
- The role of phosphate groups in the VS ribozyme–substrate interaction
- Multiple substrate binding sites in the ribozyme from Bacillus subtilis RNase P.
- Interaction of structural modules in substrate binding by the ribozyme from Bacillus subtilis RNase P.
- Peptidyl transferase centre of bacterial ribosomes: substrate specificity and binding sites.
- The evolutionary selection of DNA base pairs in gene-regulatory binding sites.