Function of P11, a tertiary base pairing in self-splicing introns of subgroup IA.
There is phylogenetic evidence for the existence of a new pairing in subgroup IA1 self-splicing introns. This tertiary interaction, called P11, which is extraneous to the catalytic centre of these ribozymes was modelled after a "pseudoknot" and grafted by computer modelling on the common core structure of group I introns that was recently proposed by Michel & Westhof. In order to probe the function of the P11 pairing, we mutated the P11 helix in the intron of the large ribosomal precursor of Saccharomyces cerevisiae mitochondria (Sc.LSU). Our experimental data show that the P11 pairing plays a role in stabilizing the overall fold of the RNA molecule. While P11 is not essential for self-splicing activity in vitro, mutants with disrupted P11 require higher concentration of MgCl2 for self-splicing. By contrast, mutants with a reinforced P11 pairing (via introduction of several G.C base-pairs) self-splice more efficiently than the wild-type at 55 degrees C. Based on this work, the possible engineering of new stable versions of the ribozyme is discussed.
Jaeger
L
L
Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
Westhof
E
E
Michel
F
F
D001482:Base Composition / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D007438:Introns / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature
D004272:DNA, Mitochondrial; D004275:DNA, Ribosomal; D046911:Macromolecular Substances; D016337:RNA, Catalytic; D015636:Magnesium Chloride