2'-Hydroxyl groups important for exon polymerization and reverse exon ligation reactions catalyzed by a group I ribozyme

Abstract

The functional importance of ribose moieties in both exons and in intron sequences proximal to the 3' splice site of a group I intron has been analyzed using a novel exon polymerization reaction. The ribozyme is a modified version of a self-splicing bacterial tRNA intron (I) that attacks a 20-nucleotide synthetic ligated exon substrate (E1.E2), yielding E1 and I.E2 by reverse exon ligation. A series of repetitive reactions then polymerize E2 on the 3' end of the intron; attack by E1 subsequently generates E1.(E2)n. Systematic deoxyribonucleotide substitution within E1.E2 was used to probe the function of 2'-hydroxyl groups in each exon and the 3'-terminal nucleotides of the intron. We find that ribose at the splice junction (U-1) and at the two adjacent positions with E1 (A-2, C-3) is important for reverse exon ligation. Within E2, deletion of 2'-hydroxyl groups of the nucleotides that form P10 does not affect reactivity. In contrast, ribose at the 3' end of the intron is essential for reverse exon ligation, and the presence of a 2'-OH group in each of the nucleotides comprising P9.0[3'] contributes to reaction efficiency. These results support a model in which specific 2'-hydroxyl groups at and adjacent to the reaction sites form tertiary contacts that serve to stabilize interactions with the catalytic core of the ribozyme. Furthermore, they suggest that the mechanism by which guanosine at the 3' end of the intron is activated for reverse exon ligation is the same as that by which guanosine mononucleotide is activated in the first step of splicing.

Novel system for analysis of group I 3' splice site reactions based on functionaltrans-interaction of the P1/P10 reaction helix with the ribozyme's catalytic core

“…In the work presented here, we report several new and significant findings that extend previously reported results obtained using the Azoarcus exon polymerization reaction (8,28). First, we have used mutational analysis to verify that the exon polymerization reaction is a faithful reporter of group I 3' splice site reactions; indeed it appears to be more sensitive to structural changes in the reacting RNA species than the simple reverse exon ligation reaction (this work and ref.…”

Molecular Recognition in a Trans Excision-Splicing Ribozyme: Non-Watson−Crick Base Pairs at the 5‘ Splice Site and ωG at the 3‘ Splice Site Can Play a Role in Determining the Binding Register of Reaction Substrates

“…For example, the contribution of tertiary interactions by the 5′ splice site u-G wobble is different between introns, as the contribution to the P. carinii ribozyme is greater than those for Tetrahymena (34) and Candida albicans (35). Moreover, 2′-hydroxyl groups from the ribose sugar units have been shown to take part in binding of Tetrahymena substrates (36)(37)(38)(39)(40), yet these interactions do not appear to be critical for P. carinii-derived ribozymes (5). Therefore, identifying and understanding the sequence requirements of the 5′ splice site for P. carinii ribozymes is useful for developing effective and specific TES ribozymes.…”