Reconstitution of a group I intron self-splicing reaction with an activator RNA.
The self-splicing rRNA intron of Tetrahymena thermophila belongs to a subgroup of group I introns that contain a conserved extra stem-loop structure termed P5abc. A Tetrahymena mutant precursor RNA lacking this P5abc is splicing-defective under standard conditions (5 mM MgCl2/200 mM NH4Cl, pH 7.5) in vitro. However, the mutant precursor RNA by itself is capable of performing the self-splicing reaction without P5abc under different conditions (15 mM MgCl2/2 mM spermidine, pH 7.5). We have investigated the functional role of the P5abc in the mechanism of the self-splicing reaction. When an RNA consisting of the P5abc but lacking the rest of the Tetrahymena intron is incubated with the mutant precursor, the self-splicing reaction proceeds highly efficiently under standard conditions (5 mM MgCl2/200 mM NH4Cl, pH 7.5). Two steps of the bimolecular self-splicing reaction can be performed accurately by a shortened precursor RNA containing all essential components required in the self-splicing reaction and an activator RNA consisting of the P5abc. Gel-mobility-shift assays suggest that two molecules associate by a direct RNA-RNA interaction during the splicing reaction. The results imply that there might exist other small RNAs whose role is to activate ribozymes.
van der Horst
G
G
Salk Institute for Biological Studies, La Jolla, CA 92037.
Christian
A
A
Inoue
T
T
D000818:Animals; D001483:Base Sequence; D007438:Introns; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D012335:RNA, Ribosomal / Q000235:genetics*; D013768:Tetrahymena / Q000235:genetics*
D015345:Oligonucleotide Probes; D012322:RNA Precursors; D012335:RNA, Ribosomal