Conformational switches involved in orchestrating the successive steps of group I RNA splicing.
Group I introns possess a conserved guanosine residue at their 3' end, termed omegaG, that, in the case of the Tetrahymena pre-rRNA, is a major determinant of the second step of splicing. We examined the role of omegaG 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 omega 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 omega residue. This suggests that a major role of omegaG 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 the splicing. The first of these is the exchange of the exogenous nucleoside for the omega nucleoside. This is the equilibrium that we can perturb by mutations at either the omega 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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BL
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215, USA.
Cech
T R
TR
D017033:Anabaena / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006151:Guanosine / Q000737:chemistry / Q000378:metabolism; D007438:Introns / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D012356:RNA, Transfer, Leu / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D012322:RNA Precursors; D012356:RNA, Transfer, Leu; D006151:Guanosine