Nucleic Acids Research Advance Access originally published online on October 31, 2008
Nucleic Acids Research 2008 36(21):6934-6943; doi:10.1093/nar/gkn824
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Nucleic Acids Research, 2008, Vol. 36, No. 21 6934-6943
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Coordination of two sequential ester-transfer reactions: exogenous guanosine binding promotes the subsequent 
G binding to a group I intron
State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China
*To whom correspondence should be addressed. Tel: +86 27 68756207; Fax: +86 27 68754945; Email: yizhang{at}whu.edu.cn
Received April 16, 2008. Revised October 14, 2008. Accepted October 14, 2008.
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 
G binding by assaying the ribozyme activity in the second reaction. We showed that adding GTP simultaneously with and prior to the esterified G in a substrate strongly accelerated the second reaction, suggesting that the early binding of GTP facilitates the subsequent binding of G. 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 G 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 G for bound GTP.
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