Dissecting and Analyzing the Secondary Structure Domains of Group I Introns through the use of Chimeric Intron Constructs

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The mitochondrial genes of the yeastSaccharomyces cerevisiaeare often interrupted by introns defined as either group I or group II. Some of the introns contained within the precursor RNAs of these genes will self splicein vitro. The fourth introns of apocytochromeb(bi4) and cytochrome oxidase (ai4) are group I introns that do not self splicein vitroeven though they can fold into the same RNA secondary structures that are characteristic of the self-splicing introns. They require an intron-encoded maturase protein and a nuclear-encoded protein (a tRNALeusynthetase) for splicingin vivo. We have divided these introns into several sequence or structural elements and assayed them individually for their ability to support self-splicing activity, This was done by replacing the equivalent elements from the self-splicing intron fromTetrahymena thermophilawith the mitochondrial elements. These intron chimeras show that peripheral sequences and the elements that define the splice sites are adequate for self-splicing but that the central portions containing the catalytic cores of ai4 and bi4 are deficient; these cores are the likely targets of the splicing proteins. In addition, the catalytic activity of theTetrahymenaintron is remarkably resistant to the structural alterations that we have introduced; this suggests that this technique will be of general utility for studying the structural and functional relationships of elements contained within different RNAs.