Conformational Switches Involved in Orchestrating the Successive Steps of Group I RNA Splicing

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Abstract
Group I introns possess a conserved guanosine residue at their 3‘ end, termed ωG, that, in the case of the Tetrahymena pre-rRNA, is a major determinant of the second step of splicing. We examined the role of ωG in self-splicing of the 249-residue group I intron of the Anabaena PCC7120 tRNAleu precursor. Contrary to observations with the Tetrahymena pre-rRNA intron, a mutation that places an adenosine residue at the ω position did not have a severe effect on the second step of splicing; neither 3‘ splice-site selection nor the rate of the second step was altered. The first step of splicing, however, was now readily reversed. This unexpected effect also resulted from a mutation that altered the nucleoside specificity of the intronic guanosine-binding site. The theme common to these mutations is that reversal of the first step of splicing results when there is not a strong interaction between the guanosine-binding site and the ω residue. This suggests that a major role of ωG is to compete with the exogenous guanosine molecule added to the intron in the first step of splicing for the single guanosine-binding site of the intron. From these data, we are able to extend the mechanism for the self-splicing reaction of this intron by proposing two distinct conformational changes between the first and second steps of splicing. The first of these is the exchange of the exogenous nucleoside for the ω nucleoside. This is the equilibrium that we can perturb by mutations at either the ω position or the guanosine-binding site. An additional conformational change then fully activates the intron for the second step of splicing.

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