Characterization of the guanosine binding process of the Twort group I ribozyme
Abstract
The ribozyme derived from bacteriophage Twort group I intron orf142-I2 undergoes RNA-cleavage reaction using a guanosine nucleophile. Comparative analysis of kinetic and thermodynamic properties of the Twort ribozyme reaction revealed that while this ribozyme shares the conserved chemical mechanism, the features related to substrate and guanosine binding differ from those of other group I ribozymes. First, kinetic analysis under the ( kcat/Km)S condition suggests that the length of P1 helix of the Twort ribozyme is a basis of its (kcat/Km)S value and rate-limiting step for (kcat/ Km)S. These are similar to those of the Tetrahymena ribozyme, rather than those of the Anabaena and Azoarcus ribozymes. Second, guanosine binding of the Twort ribozyme was much tighter than for the Anabaena and Tetrahymena ribozymes. Furthermore, the thermodynamic values were different from those of other ribozymes. To explain the differences in the guanosine-binding process, we hypothesize that the unbound conformations of the guanosine-binding site differ: that of the Twort ribozyme is more like the guanosine-bound conformation while those of the other group I introns are more collapsed. This hypothesis is supported by the presence of the P7.1-7.2 subdomain that stabilizes the conformation of the active site. In contrast to the non-conserved thermodynamics of the guanosine-binding process, comparison of the highly conserved guanosine-bound structures allowed us to investigate how guanosine binding organizes the active site. Superimposition of the guanosine- and deoxyguanosine-bound structures demonstrated that their active site show different conformations, and this may be due to the hydrogen bonds between the 2'-OH of G and the 2'-OH of A119. Kinetic study with the mutant lacking the 2'-OH of A119 suggested that these interactions are involved in tight binding of guanosine and organization of the catalytically active site. Neurospora mt tyrosyl-tRNA synthetase (CYT-18) binds the Twort ribozyme by recognizing conserved features of group I introns. Determination of Mg2+ dependence of the guanosine binding affinity and catalytic rate of the CYT-18-assisted ribozyme reaction demonstrated that CYT-18 stabilizes the tertiary structure of the guanosine-binding site, and its binding minimizes the contribution of structural functions of Mg2+ in catalysis. This system may allow probing of the roles of the catalytic Mg2+ ions.
Degree
Ph.D.
Advisors
Golden, Purdue University.
Subject Area
Molecular biology|Biophysics
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