Nucleic Acids Research, 2000, Vol. 28, No. 3 720-727
© 2000 Oxford University Press
Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage
Medizinische Universität zu Lübeck, Institut für Biochemie, Ratzeburger Allee 160, D-23538 Lübeck, Germany and 1Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology and Department of Chemistry, University of Chicago, 5841 South Maryland Avenue, MC1028, Chicago, IL 60637, USA
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.
* To whom correspondence should be addressed. Tel: +45 1 500 4065; Fax: +45 1 500 4068; Email: hartmann@biochem.mu-luebeck.de Present addresses: Jens M. Warnecke, Universität Witten/Herdecke, Institut für Molekularbiologie, Stockumer Straße 10, D-58453 Witten, Germany Erik J. Sontheimer, Northwestern University, Department of Biochemistry, Molecular Biology and Cell Biology, 2153 North Campus Drive, Evanston, IL 60208, USA
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Q. Vicens, P. J. Paukstelis, E. Westhof, A. M. Lambowitz, and T. R. Cech Toward predicting self-splicing and protein-facilitated splicing of group I introns RNA, October 1, 2008; 14(10): 2013 - 2029. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. V. Kazantsev, A. A. Krivenko, D. J. Harrington, S. R. Holbrook, P. D. Adams, and N. R. Pace Crystal structure of a bacterial ribonuclease P RNA PNAS, September 20, 2005; 102(38): 13392 - 13397. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Cuzic and R. K. Hartmann Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor Nucleic Acids Res., May 2, 2005; 33(8): 2464 - 2474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-G. Ren, L. A. Kirsebom, and A. Virtanen Coordination of Divalent Metal Ions in the Active Site of Poly(A)-specific Ribonuclease J. Biol. Chem., November 19, 2004; 279(47): 48702 - 48706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Sabbagh, K. J. Fettes, R. Gosain, I. A. O'Neil, and R. Cosstick 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 Nucleic Acids Res., January 23, 2004; 32(2): 495 - 501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Persson, S. Cuzic, and R. K. Hartmann Catalysis by RNase P RNA: UNIQUE FEATURES AND UNPRECEDENTED ACTIVE SITE PLASTICITY J. Biol. Chem., October 31, 2003; 278(44): 43394 - 43401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Takagi, M. Warashina, W. J. Stec, K. Yoshinari, and K. Taira SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes Nucleic Acids Res., May 1, 2001; 29(9): 1815 - 1834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yoshinari and K. Taira A further investigation and reappraisal of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme Nucleic Acids Res., April 15, 2000; 28(8): 1730 - 1742. [Abstract] [Full Text] [PDF]
|
|