Role of the 2'-Hydroxyl at the 3'-Splice Site in the Second Step of the Group II Ribozyme Self-splicing Reaction
Vereeke, Kelly M.
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The removal of introns from pre-mRNA is a crucial aspect of eukaryotic gene expression. Most introns are removed by the spliceosome, a large ribonucleoprotein complex. However, a small subset of introns (group II) are autocatalytic and self-splice in the absence of protein factors. Group II intron splicing occurs via two consecutive phosphotransesterification reactions. The first step generates a free 5' exon and intron/3' exon intermediate. In the second step, the 3'-hydroxyl of the 5' exon serves as a nucleophile to attack the 3' splice site. This step generates ligated exons and liberates the intron. The substitution of the 2' -hydroxyl group at the 3' splice site with a hydrogen was previously demonstrated to strongly inhibit the exon ligation step of splicing. Possible roles for this 2' -hydroxyl include: acceptance of a hydrogen bond, donation of a hydrogen bond, and/or coordination to a metal ion. In this thesis, we tested these models by examining how different modifications at the 2' position (2'-fluoro, 2'-0-methyl, 2'amino) influenced the exon ligation step. Replacement of the 2'-hydroxyl with 2'-0- methyl and 2'-fluorine, both of which can accept, but not donate, a hydrogen bond, strongly inhibited the rate of exon ligation. This result suggested that the 2' -hydroxyl does not accept a hydrogen bond. However, substitution with a 2' -amino group only moderately reduced the rate of exon ligation at a pH where the amino group is protonated and can donate, but not accept a hydrogen bond. Therefore, hydrogen bond donation by the 2'-hydroxyl may be important during the exon ligation step of splicing. Lastly, the rate of exon ligation for the 2' -amino substrate was stimulated by the addition of Mn2+, and may be indicative of a direct metal ion interaction. Further research is needed to confirm the role of the 2' -hydroxyl, which will lead to a better understanding of selfsplicing mechanisms. Splicing mechanisms are important to the overall understanding of eukaryotic gene expression and may serve as a model for a novel disease therapy.