The 2'-hydroxyl group of the guanosine nucleophile donates a functionally important hydrogen bond in the tetrahymena ribozyme reaction.
In the first step of self-splicing, group I introns utilize an exogenous guanosine nucleophile to attack the 5'-splice site. Removal of the 2'-hydroxyl of this guanosine results in a 10 (6)-fold loss in activity, indicating that this functional group plays a critical role in catalysis. Biochemical and structural data have shown that this hydroxyl group provides a ligand for one of the catalytic metal ions at the active site. However, whether this hydroxyl group also engages in hydrogen-bonding interactions remains unclear, as attempts to elaborate its function further usually disrupt the interactions with the catalytic metal ion. To address the possibility that this 2'-hydroxyl contributes to catalysis by donating a hydrogen bond, we have used an atomic mutation cycle to probe the functional importance of the guanosine 2'-hydroxyl hydrogen atom. This analysis indicates that, beyond its role as a ligand for a catalytic metal ion, the guanosine 2'-hydroxyl group donates a hydrogen bond in both the ground state and the transition state, thereby contributing to cofactor recognition and catalysis by the intron. Our findings continue an emerging theme in group I intron catalysis: the oxygen atoms at the reaction center form multidentate interactions that function as a cooperative network. The ability to delineate such networks represents a key step in dissecting the complex relationship between RNA structure and catalysis.
Hougland
James L
JL
Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.
Sengupta
Raghuvir N
RN
Dai
Qing
Q
Deb
Shirshendu K
SK
Piccirilli
Joseph A
JA
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006151:Guanosine / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D007438:Introns; D015394:Molecular Structure; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013768:Tetrahymena / Q000201:enzymology*
D016337:RNA, Catalytic; D006151:Guanosine