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

Abstract

A group I intron from a bacterial tRNA precursor has been converted into an RNA enzyme that catalyzes the efficient polymerization of oligoribonucleotide analogs of tRNA exons using a reaction scheme consisting of multiple cycles of reverse and forward exon ligation reactions. Here, we present results showing that this system represents a novel and useful tool for the analysis of 3' splice site reactions of group I ribozymes. First, analysis of variant substrates containing base substitutions in group I secondary structure elements P1, P9.0 and P10 confirms that exon polymerization is dependent on these structures, and therefore constitutes an appropriate and relevant model system for studying the exon ligation step of splicing. Second, to probe interactions between the intron's catalytic core and the bases and backbone of the P1/P10 reaction helix, two successful strategies for separating the internal guide sequence from the intron core were devised. One such strategy uses a construct in which the reaction helix interacts functionally with the catalytic core using only tertiary contacts. Further stabilization of this interaction through the inclusion of a 7 bp intermolecular P2 helix generates increased reaction efficiency. Third, when provided with two reaction helices, the ribozyme synthesizes mixed polymers through a mechanism that involves sequential binding and release of the duplexes. Fourth, in these reactions, turnover of the external guide sequence requires unwinding and annealing of the P2 helix, suggesting that P2 unwinding may occur during group I splicing. These results provide novel experimental tools to probe the relatively poorly understood 3' splice site reactions of group I introns, and may be relevant to ribozyme-catalyzed assembly and recombination of oligomers in prebiotic scenarios.

Spontaneous Cooperative Assembly of Replicative Catalytic RNA Systems

“…The trans-splicing activity of the blind Azoarcus ribozyme is in accordance with a previous study that demonstrated that if the IGS were disarticulated from the catalytic core on a separate fragment, and then an effectively full-length ribozyme could polymerize oligonucleotide sequences on its 3´ end (Chowrira et al, 1995). In that study an exogenous IGS (termed the external guide sequence, or EGS) required the GUG from P1 plus an additional 7 nt from P10 of the natural bacterial intron.…”

“…Second, it is behaving as an exogenous IGS, bringing a requisite GUG into the catalytic core of a ribozyme complex. The multifunctional fragment containing the exogenous IGS is held likely in place by tertiary interactions (Chowrira et al, 1995) such as those postulated in Figure 9. And third, it is being utilized as a substrate for recombination, in which its CAU sequence (or a lowerror variant) is targeted by an IGS for transesterification to another RNA molecule.…”

“…Notably, these enzymes often remain active when broken into non-covalent trans complexes. The work of Inoue and others has shown that the Tetrahymena, sunY, Azoarcus, Synechococcus, phage T4 td and other group I introns can retain catalytic efficiency when fragmented into several non-covalent pieces and then reconstituted in trans via base-pairing and tertiary interactions (Beaudry and Joyce, 1990;Chowrira et al, 1995;Doudna and Cech, 1995;Ikawa et al, 2000a;van der Horst et al, 1991). These studies have isolated the regions of the ribozyme that are necessary to carry out the various catalytic events of self-splicing, a multi-step process that involves two transesterification reactions plus often a hydrolytic cleavage reaction.…”

Trans-splicing with the group I intron ribozyme from Azoarcus

“…Previous studies used high Mg 2+ concentrations (10-100 mM) and high temperature (48°C-60°C, with the optimum around 60°C) (Chowrira et al 1993(Chowrira et al , 1995Tanner and Cech 1996;Kuo et al 1999;Chauhan et al 2005;Hayden et al 2005;Sinan et al 2011). Under these conditions, the Azoarcus ribozyme is able to catalyze an impressive variety of transreactions.…”

“…Under these conditions, the Azoarcus ribozyme is able to catalyze an impressive variety of transreactions. By repetitively splicing a substrate the Azoarcus ribozyme can cause an effect like RNA polymerization (Chowrira et al 1993(Chowrira et al , 1995. By trans-ligating RNA oligomers the Azoarcus ribozyme can create active ribozymes from inactive ribozyme fragments (Hayden et al 2005).…”