IN-VITRO GENETIC-ANALYSIS OF THE STRUCTURAL FEATURES OF THE PRE-TRANSFER RNA REQUIRED FOR DETERMINATION OF THE 3' SPLICE-SITE IN THE INTRONEXCISION REACTION

During processing of intron-containing pre-tRNAs, the Xenopus laevis s plicing endonuclease binds the precursor and cleaves it at both the 5' and 3' splice sites. In vitro selection was used to determine structu ral features characteristic of precursor tRNA molecules that are activ e in this reaction. We performed two types of selection, one for molec ules that are not cut, the other for molecules that are cut at only on e site. The results shed light on various aspects of the intron excisi on reaction, including the importance of the three-dimensional structu re of the mature domain for recognition and binding of the enzyme, the active role played by the single-stranded region of the intron, and t he importance of the cardinal positions which, although not necessaril y occupied by the same base in all precursors, nevertheless play a fun damental 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 posit ion 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 int ron. We discuss the role of the cardinal positions in the reaction.