An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

被引:6
|
作者
Sengupta, Raghuvir N. [1 ]
Van Schie, Sabine N. S. [1 ,2 ]
Giambasu, George [3 ]
Dai, Qing [4 ,5 ]
Yesselman, Joseph D. [1 ]
York, Darrin [3 ]
Piccirilli, Joseph A. [4 ,5 ]
Herschlag, Daniel [1 ,6 ,7 ,8 ]
机构
[1] Stanford Univ, Dept Biochem, Stanford, CA 94305 USA
[2] Leiden Univ, Leiden Inst Chem, NL-2333 CC Leiden, Netherlands
[3] Rutgers State Univ, Dept Chem & Chem Biol, Piscataway, NJ 08854 USA
[4] Univ Chicago, Dept Chem, 5735 S Ellis Ave, Chicago, IL 60637 USA
[5] Univ Chicago, Dept Biochem & Mol Biol, 920 E 58Th St, Chicago, IL 60637 USA
[6] Stanford Univ, Dept Chem Engn, Stanford, CA 94305 USA
[7] Stanford Univ, Dept Chem, Stanford, CA 94305 USA
[8] Stanford Univ, Dept Chem Engn & Med Human Hlth ChEM H, Stanford, CA 94305 USA
基金
美国国家卫生研究院;
关键词
active site; conformational change; metal ion; noncoding RNA; RNA catalysis; GUANOSINE-BINDING-SITE; INDUCED-FIT MECHANISM; METAL-ION INTERACTION; PYRENE-LABELED SUBSTRATE; PEPTIDE-BOND FORMATION; DETECTED STOPPED-FLOW; SELF-SPLICING RNA; 2 OH GROUPS; CRYSTAL-STRUCTURE; CONFORMATIONAL-CHANGE;
D O I
10.1261/rna.053710.115
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
Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E.S.G and E.G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and amino (-NH2) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E.G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both OH groups to contact a different active site metal ion, termed M-c, to make what. are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.
引用
收藏
页码:32 / 48
页数:17
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