Coordination of two sequential ester-transfer reactions: exogenous guanosine binding promotes the subsequent omegaG binding to a group I intron.
Self-splicing of group I introns is accomplished by two sequential ester-transfer reactions mediated by sequential binding of two different guanosine ligands, but it is yet unclear how the binding is coordinated at a single G-binding site. Using a three-piece trans-splicing system derived from the Candida intron, we studied the effect of the prior GTP binding on the later omegaG binding by assaying the ribozyme activity in the second reaction. We showed that adding GTP simultaneously with and prior to the esterified omegaG in a substrate strongly accelerated the second reaction, suggesting that the early binding of GTP facilitates the subsequent binding of omegaG. GTP-mediated facilitation requires C2 amino and C6 carbonyl groups on the Watson-Crick edge of the base but not the phosphate or sugar groups, suggesting that the base triple interactions between GTP and the binding site are important for the subsequent omegaG binding. Strikingly, GTP binding loosens a few local structures of the ribozyme including that adjacent to the base triple, providing structural basis for a rapid exchange of omegaG for bound GTP.
Bao
Penghui
P
Department of Biochemistry and Molecular Biology, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Wu
Qi-Jia
QJ
Yin
Ping
P
Jiang
Yanfei
Y
Wang
Xu
X
Xie
Mao-Hua
MH
Sun
Tao
T
Huang
Lin
L
Mo
Ding-Ding
DD
Zhang
Yi
Y
D001665:Binding Sites; D002175:Candida / Q000201:enzymology / Q000235:genetics; D004952:Esters / Q000737:chemistry; D005091:Exons; D006151:Guanosine / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D007438:Introns; D007700:Kinetics; D022821:RNA Splice Sites; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism
D004952:Esters; D022821:RNA Splice Sites; D016337:RNA, Catalytic; D006151:Guanosine; D006160:Guanosine Triphosphate