Ionization of a critical adenosine residue in the Neurospora Varkud Satellite ribozyme active site

被引:52
作者
Jones, FD
Strobel, SA
机构
[1] Yale Univ, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA
[2] Yale Univ, Dept Chem, New Haven, CT 06520 USA
关键词
D O I
10.1021/bi020707t
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Varkud Satellite (VS) ribozyme catalyzes a site-specific self-cleavage reaction that generates 5'-OH and 2',3'-cyclic phosphate products. Other ribozymes that perform an equivalent reaction appear to employ ionization of an active site residue, either to neutralize the negatively charged transition state or to act as a general acid-base catalyst. To test for important base ionization events in the VS ribozyme ligation reaction, we performed nucleotide analogue interference mapping (NAIM) with a series of ionization-sensitive adenosine and cytidine analogues. A756, a catalytically critical residue located within the VS active site, was the only nucleotide throughout the VS ribozyme that displayed the pH-dependent interference pattern characteristic of functional base ionization. We observed unique rescue of 8-azaadenosine (pK(a) 2.2) and purine riboside (pK(a) 2.1) interference at A756 at reduced reaction pH, suggestive of an ionization-specific effect. These results are consistent with protonation and/or deprotonation of A756 playing a direct role in the VS ribozyme reaction mechanism. In addition, NAIM experiments identified several functional groups within the RNA that play important roles in ribozyme folding and/or catalysis. These include residues in helix II, helix VI (730 loop), the II-III-VI and III-IV-V helix junctions, and loop V.
引用
收藏
页码:4265 / 4276
页数:12
相关论文
共 48 条
[1]   Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme [J].
Andersen, AA ;
Collins, RA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (14) :7730-7735
[2]   Rearrangement of a stable RNA secondary structure during VS ribozyme catalysis [J].
Andersen, AA ;
Collins, RA .
MOLECULAR CELL, 2000, 5 (03) :469-478
[3]   The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution [J].
Ban, N ;
Nissen, P ;
Hansen, J ;
Moore, PB ;
Steitz, TA .
SCIENCE, 2000, 289 (5481) :905-920
[4]   Identification of functional domains in the self-cleaving Neurospora VS ribozyme using damage selection [J].
Beattie, TL ;
Collins, RA .
JOURNAL OF MOLECULAR BIOLOGY, 1997, 267 (04) :830-840
[5]   Solution structure of loop a from the hairpin ribozyme from tobacco ringspot virus satellite [J].
Cai, ZP ;
Tinoco, I .
BIOCHEMISTRY, 1996, 35 (19) :6026-6036
[6]   REACTION CONDITIONS AND KINETICS OF SELF-CLEAVAGE OF A RIBOZYME DERIVED FROM NEUROSPORA VS RNA [J].
COLLINS, RA ;
OLIVE, JE .
BIOCHEMISTRY, 1993, 32 (11) :2795-2799
[7]   Two decades of RNA catalysis [J].
DeRose, VJ .
CHEMISTRY & BIOLOGY, 2002, 9 (09) :961-969
[8]  
Fasman G. D., 1975, HDB BIOCH MOL BIOL
[9]   Crystal structure of a hepatitis delta virus ribozyme [J].
Ferré-D'Amaré, AR ;
Zhou, KH ;
Doudna, JA .
NATURE, 1998, 395 (6702) :567-574
[10]   A pH controlled conformational switch in the cleavage site of the VS ribozyme substrate RNA [J].
Flinders, J ;
Dieckmann, T .
JOURNAL OF MOLECULAR BIOLOGY, 2001, 308 (04) :665-679