On how hydrolysis at the 3′ end is prevented in the splicing of a sequentially folded group I intron

Abstract

On how hydrolysis at the 3' end is prevented in the splicing of a sequentially folded group I intron WC propose a dynamic model for the compelition bctwcen exon-exon ligation and 3'-end hydrolysis valid for sequentially folded pre-mRNA introns of group I. This model accounts for the delay in the formation of conserved helix PlO until the 5' exon has been cleaved. a requirement to prevent hydrolysis at the 3' end of the intron. The model is rooted on computer simulations whereby the pre-mRNA searches for its structure as it is being transcribed. Thus, a competing interaction, engging the internal guiding sequence. occurs initially and prevents PlO from forming until the 3'end of the 5' exon is habilitated as a nuclcophilic agent. It is further shown that a destabilization of the compeling interaction invariably leads to 3 hydrolysis, crippling the splicing capability of the intron. The results may be probed by sitedirected mutagen&s.Ribozyme; 3' l-iydrolysis; In vivo RNA folding I. INTRODUCTIONPerhaps the most important reaction competing with exon ligation in the splicing of group I introns is hydrolysis at the 3' end of the intron. The prevention of this competing reaction poses a major difficulty to experimentalists attempting to engineer splicing conditions. Hydrolysis might be precluded if the formation of the conserved helix PlO is delayed until 5' cleavage had taken place and the nucleophilic 3' end of the already spliced 5' exon is ready to attack the 3' intronexon junction [l]. Only then PI0 should be allowed to foml. We shall verify this scenario by simulating the folding of the fourth intron of the yeast apocytochrome b gene (YCOB4) [2,3], an intron that requires trunsacting factors to fold into an active structure and overcome its structural deficiencies.The chronological order of events demands a dynamical model of folding where interaction I' engages the internal guiding sequence (IGS) until 5' cleavage disrupts it, precluding the premature formation of PI0 (see Fig. 1). Thus, the postponement in the formation of PI0 finds concrete meaning in a sequential model for the folding of the pre-mRNA under scrutiny. We advocate that the structure is searched concurrently with the assembling of the intron. In other words, folding is concomitant with transcription and results from the Published by Ehv/er Scicncr Publishers B. V.

Enhancing the Second Step of the Trans Excision-Splicing Reaction of a Group I Ribozyme by Exploiting P9.0 and P10 for Intermolecular Recognition

“…Evidently, similar to that seen in the self-splicing reaction (11)(12)(13)(14), P1ex and P10 are dynamic and compete with one another during each step of the TES reaction. Note, however, that in our studies lacking RE3, in which case both P10 and P1ex are not permitted, the second step of the TES reaction (only) goes poorly.…”

“…This 5‘ C could form a Watson−Crick base pair with the 3‘ G of RE3, which could result in the formation of an extended P1 helix (called P1ex). Such P1ex helices have been shown to be inhibitory in other contexts with group I intron-based catalytic RNAs ( − ).…”

“…The Bridging Region of the Substrate Can Inhibit the TES Reaction by Forming a P1ex Helix. In accordance with the presupposition that the 5‘ end of the excised region of TES substrates can base pair with RE3 and compete with the 3‘ exon for binding RE3 ( − ), we analyzed a series of modified ribozymes that are expected to form stability-enhanced P1ex or P10 helices when paired with the 36-mer substrate . By modulating the strength of these base-pairing elements, we can deduce whether TES inhibition likely results from P1ex formation interfering with P10 formation.…”

Localization of strain in the RNA backbone and its functional implication

“…As an illustration, the results are specialized for the species YCOB5 (TV =738), the fifth intron of the apocytochrome b gene of yeast [11,12], where the concensus structure has been established since this species belongs to the so-called group-la intron family [2]. Only 15% of the residues are conserved for chains of length comparable to that of the YCOB5.…”

“…The substructure which could potentially compete with the concensus P10 interaction [12] does not occur at the final stage of formation of the secondary structure, from which the constraint ft is derived.…”