A group II self-splicing intron from the brown alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation.
We have investigated the reactivity of three of the seven group II introns encoded by the mitochondrial genome of the brown alga Pylaiella littoralis. While the first intron in the protein-coding cox1 gene could not be induced to self-splice under any of the conditions tested, the first two introns in the gene encoding the large ribosomal subunit are reactive in vitro and splice primarily by the standard group II two-step transesterification pathway. Intron 2 proved to be of exceptional interest, because in contrast to all group II molecules known so far, its optimal magnesium concentration is less than 10 mM and it still carries out accurate splicing at concentrations as low as 0.1 mM magnesium. Analysis of reaction products under optimal conditions showed no evidence of hydrolysis at the 5' splice site and up to 90% of precursor molecules could be converted into excised lariat intron, which migrated as a single band on non-denaturing polyacrylamide gels. Absorbance versus temperature profiles generated from the lariat form of intron 2 reveal the existence of an early melting component, the amplitude of which does not depend on the way the molecules were purified, i.e. with or without a denaturation step. This highly cooperative transition, whose position along the temperature axis changes with the concentration of magnesium, is proposed to consist of the unfolding of the tertiary structure of the molecule. We conclude that group II introns, which are the largest known ribozymes, can form conformationally homogeneous populations of molecules suitable for physical-chemical studies of higher-order structure.
Costa
M
M
Centre de Génétique Moléculaire du C.N.R.S., 91190 Gif-sur-Yvette, France.
Fontaine
J M
JM
Loiseaux-de Goër
S
S
Michel
F
F
D003576:Electron Transport Complex IV / Q000235:genetics; D007438:Introns; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D000459:Phaeophyceae / Q000235:genetics*; D012326:RNA Splicing; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics
D012335:RNA, Ribosomal; D003576:Electron Transport Complex IV; D008274:Magnesium