Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3′-S-phosphorothiolate internucleotide linkage
AUTOR(ES)
Warnecke, Jens M.
FONTE
Oxford University Press
RESUMO
Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3′-OH and 5′-phosphate termini, and at least two metal ions are thought to be essential for catalysis. To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3′-oxyanion leaving group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3′-S-phosphorothiolate linkages at the RNase P cleavage site were synthesized. With a 3′-S-phosphorothiolate-modified ptRNA carrying a 7 nt 5′-flank, a complete shift of the cleavage site to the next unmodified phosphodiester in the 5′-direction was observed. Cleavage at the modified linkage was not restored in the presence of thiophilic metal ions, such as Mn2+ or Cd2+. To suppress aberrant cleavage, we also constructed a 3′-S-phosphorothiolate-modified ptRNA with a 1 nt 5′-flank. No detectable cleavage of this substrate was seen in reactions catalyzed by RNase P RNAs from Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions. Ground state binding of modified ptRNAs was not impaired, suggesting that the 3′-S-phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=102553Documentos Relacionados
- Nucleotides in precursor tRNAs that are required intact for catalysis by RNase P RNAs.
- Synthesis of phosphorothioamidites derived from 3′-thio-3′-deoxythymidine and 3′-thio-2′,3′-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3′-S-phosphorothiolate linkage
- Effects of tRNA-intron structure on cleavage of precursor tRNAs by RNase P from Saccharomyces cerevisiae.
- Rpp2, an essential protein subunit of nuclear RNase P, is required for processing of precursor tRNAs and 35S precursor rRNA in Saccharomyces cerevisiae
- Ribosome binding by tRNAs with fluorescent labeled 3' termini.
