In vitro genetic analysis of the structural features of the pre‐tRNA required for determination of the 3′ splice site in the intron excision reaction.

During processing of intron‐containing pre‐tRNAs, the Xenopus laevis splicing endonuclease binds the precursor and cleaves it at both the 5′ and 3′ splice sites. In vitro selection was used to determine structural features characteristic of precursor tRNA molecules that are active in this reaction. We performed two types of selection, one for molecules that are not cut, the other for molecules that are cut at only one site. The results shed light on various aspects of the intron excision reaction, including the importance of the three‐dimensional structure of the mature domain for recognition and binding of the enzyme, the active role played by the single‐stranded region of the intron, and the importance of the cardinal positions which, although not necessarily occupied by the same base in all precursors, nevertheless play a fundamental role in the splicing reaction. A precursor can be cut at the 3′ site if a base in the single‐stranded loop of the intron is allowed to pair (A‐I pair) with the base of the 5′ exon situated at the position immediately following the anticodon stem [first cardinal position (CP1)]. The nature of the bases involved in the A‐I pair is important, as is the position of the base in the single‐stranded loop of the intron. We discuss the role of the cardinal positions in the reaction.