Structure and conformational rearrangements during splicing of the ribozyme component of group II introns

Abstract : Group II introns are a class of RNAs best known for their ribozyme-catalyzed, self-splicing reaction. Under certain conditions, the introns can excise themselves from precursor mRNAs and ligate together their flanking exons, without the aid of proteins. Group II introns generally excise from pre-mRNA as a lariat, like the one formed by spliceosomal introns, similarities in the splicing mechanism suggest that group II introns and nuclear spliceosomal introns may share a common evolutionary ancestor.Despite their very diverse primary sequences, group II introns are defined by a highly conserved secondary structure. This generally consists of six domains (Domain I-Domain VI; D1-D6) radiating from a central wheel. Each of the six intronic domains has a specific role in folding, conformational rearrangements or catalysis. The native conformation of a group II intron is sustained by intra- and interdomain long-range tertiary interactions, which are critical either for folding of the intron to the native state or for its catalytic activity. In brief, Domain V interacts with Domain I to form the minimal catalytic core; Domain VI contains a highly conserved bulged adenosine serving as the branch-point nucleotide. DII and Domain III contribute to RNA folding and catalytic efficiency. Domain IV, which encodes the intron ORF, is dispensable for ribozyme activity.Group II intron splicing proceeds through two step transesterification reactions which yield ligated exons and an excised intron lariat. It is initiated by the 2'-hydroxyl group of the bulged adenosine within Domain 6, which serves as a branch point and attacks the phosphate at the 5'-end of the intron, thus releasing the 5'-exon while forming a lariat structure in the first step. The released 5'-exon, which is bound to the intron through base pairing interactions, is then positioned correctly to attack the 3'-splice site with its free 3'-OH in the second step of splicing. It is generally believed that the structure of a group II ribozyme undergoes conformational rearrangements between first step and second step and domain VI must play a central role in the process. However, despite the identification of several interdomain tertiary interactions, neither NMR nor chemical probing studies have been successful in determining the local surroundings of the branch-point adenosine and neighboring domain VI nucleotides in the ribozyme active site. By using phylogenetic analysis and molecular modelling, we have identified several areas of the molecule which have the potential to constitute the docking site of domain VI. Mutations were introduced in putative binding sites and the resulting, mutant RNAs have been kinetically characterized. This has allowed us to identify a site within the ribozyme that appears to be specifically involved in the branching reaction. In order to further investigate the interaction between that site and domain VI, we set up a system in which the docking of domain VI into its presumed binding site is ensured by the addition of DNA/RNA oligos that position the two RNA elements in an appropriate orientation. By combining the information from such experiments, we have built an atomic-resolution model of the complex formed by domain VI, the branch site and the rest of the intron at the time at which splicing is initiated.
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Agricultural sciences. Université Paris Sud - Paris XI; National Tsing-Hua University, 2011. English. <NNT : 2011PA112093>
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Submitted on : Monday, January 16, 2012 - 5:47:44 PM
Last modification on : Tuesday, January 17, 2012 - 9:31:55 AM




  • HAL Id : tel-00660480, version 1



Cheng-Fang Li. Structure and conformational rearrangements during splicing of the ribozyme component of group II introns. Agricultural sciences. Université Paris Sud - Paris XI; National Tsing-Hua University, 2011. English. <NNT : 2011PA112093>. <tel-00660480>




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