PubMed · 9739093
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M Holford, T W Muir. 1998-08-15. Adding 'splice' to protein engineering.. https://doi.org/10.1016/s0969-2126(98)00097-5
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Group I, group II and spliceosomal introns splice by two sequential transesterification reactions. For both spliceosomal and group II introns, the first-step reaction occurs by nucleophilic attack on the 5' splice junction by the 2' hydroxyl of an internal adenosine, forming a 2'-5' phosphodiester branch in the intron. The second reaction joins the two exons with a 3'-5' phosphodiester bond and releases intron lariat. In vitro, group II introns can self-splice by an efficient alternative pathway in which the first-step reaction occurs by hydrolysis. The resulting linear splicing intermediate participates in normal second-step reactions, forming spliced exon and linear intron RNAs. Here we show that the group II intron first-step hydrolysis reaction occurs in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations. As expected, the mutations block branching, but surprisingly still allow accurate splicing. This hydrolysis pathway may have been a step in the evolution of splicing mechanisms.
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Two temperature-conditional secretory mutations, sec1 and sec5, cause the accumulation of post-Golgi vesicles when strains containing these mutations are grown at 37 degrees C. In addition to accumulating vesicles, the mutants do not esterify free sterol on rich media at the restrictive temperature. It is the high level of inositol in the media that causes this condition in the yeast Saccharomyces cerevisiae, not a defective steryl ester synthase or lack of substrates. When strains containing the sec1 or sec5 mutation were transformed separately with a plasmid carrying SEC1 and SEC5, the esterification and secretory defects were alleviated. Double mutants containing sec6, sec14 or sec18 with either a sec1 or sec5 mutation have normal esterification levels. Strains with suppressor mutations were isolated that grew at 37 degrees C, esterified sterols and had diminished accumulation of vesicles, when grown at the restrictive temperature on defined media with additional inositol. Electron microscopy was used to examine vesicle accumulation, the number of lipid droplets, and to further characterize the esterification defect. When grown at 37 degrees C on defined medium, the strains with sec5 or sec1 accumulated the usual secretory vesicles, but when grown under similar conditions with elevated levels of inositol, accumulated an additional vesicular-like body.