Poster abstracts

Poster number 34 submitted by Hajeong Kim

Biochemical and thermodynamic characterization of guanosine substrate-binding of a group I Intron from bacteriophage Twort

Hajeong Kim (Biochemistry, Purdue university), Josh Sokoloski (Chemistry, Penn State University), Philip C Bevilacqua (Chemistry, Penn State University), Barbara Golden (Biochemistry, Purdue university)

Abstract:
Binding of guanosine substrate to the active site of group I intron has been investigated by kinetic and thermodynamic tools. Surprisingly, this intron binds guanosine very tightly, much tighter than the Anabaena and Tetrahymena group I introns. To explore why this intron might bind guanosine so tightly, we characterized the thermodynamics of G-binding by isothermal titration calorimetry. Guanosine binding by the Twort intron is enthalpically favored, contrasted by entropically favored guanosine binding with delta H =~0 in the other group I introns. To explain these differences, we hypothesize that the unbound conformation of the Twort ribozyme guanosine binding site more closely resembles bound one, which may be due at least in part to the presence of P7.1-7.2 subdomain.

We have also explored features of substrate recognition that are common to all group I introns. Recent crystallographic studies of the deoxyguanosine- and guanosine-bound group I intron ribozyme demonstrated that 2'-OH of guanosine substrate makes hydrogen bonds to 2'-OH of A119 (Golden et al., 2005; Adams et al., 2004). We hypothesize that this interaction helps to organize the structure of the active site. To test this, we generated a mutant ribozyme (Tw-dA119) having 2'-H substitution on A119, and determined the dissociation constant for guanosine of Tw-dA119. The Tw-dA119 bound at least 200-fold less tightly to guanosine, indicating that the active site of the ribozyme is not well organized without 2'-OH of A119. This is in agreement with crystal structures of group I introns which reveal the differences between guanosine-bound and deoxyguanosine-bound active site conformation and the inability of deoxyguanosine to serve as a substrate.

References:
Adams PL, Stahley MR, Kosek AB, Wang J, Strobel SA. 2004.Crystal structure of a self-splicing group I intron with both exons. Nature. 430:45-50.
Golden BL, Kim H, Chase E. 2005. Crystal structure of a phage Twort group I ribozyme-product complex. Nat Struct Mol Biol. 12:82-9.

Keywords: Group I intron, ribozymes, thermodynamics