Dissecting and analyzing the secondary structure domains of group I introns through the use of chimeric intron constructs.
The mitochondrial genes of the yeast Saccharomyces cerevisiae are 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 splice in vitro. The fourth introns of apocytochrome b (bi4) and cytochrome oxidase (ai4) are group I introns that do not self splice in vitro, even 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 tRNALeu synthetase) for splicing in 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 from Tetrahymena thermophila with the mitochondrial elements. These intron chimeras show that peripheral sequences and the elements that define the splice sites are adequate for self-splicing activity 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 the Tetrahymena intron 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.