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Six RNA aptamers that bind to yeast phenylalanine tRNA were identified by in vitro selection from a random-sequence pool. The two most abundantly represented aptamers interact with the tRNA anticodon loop, each through a sequence block with perfect Watson-Crick complementarity to the loop. It was possible to truncate one of these aptamers to a simple hairpin loop that forms a classical 'kissing complex' with the anticodon loop. Three other aptamers have nearly complete complementarity to the anticodon loop. The sixth aptamer has two sequence blocks, one complementary to the tRNA T loop and the other to the D loop; this aptamer binds better to a mutant tRNA that disrupts the normal D-loop/T-loop tertiary interaction than to the wild-type tRNA. Selection of complements to tRNA loops occurred despite an attempt to direct binding to tertiary structural features of tRNA. This serves as a reminder of how special the RNA-RNA interactions are that are not based on complementarity. Nonetheless, these aptamers must present the tRNA complement in some special structural context; the simple single-strand complement of the anticodon loop did not bind tRNA effectively.

Saccharomyces cerevisiae uses G protein-coupled receptors for signal transduction. We show that a fusion protein between the alpha-factor receptor (Ste2) and the Galpha subunit (Gpa1) transduces the signal efficiently in yeast cells devoid of the endogeneous STE2 and GPA1 genes. To evaluate the function of different domains of Galpha, a chimera between the N-terminal region of yeast Gpa1 and the C-terminal region of rat Gsalpha has been constructed. This chimeric Gpa1-Gsalpha is capable of restoring viability to haploid gpa1Delta cells, but signal transduction is prevented. This is consistent with evidence showing that the C-terminus of the homologous Galpha is required for receptor-G protein recognition. Surprisingly, a fusion protein between Ste2 and Gpa1-Gsalpha is able to transduce the signal efficiently. It appears, therefore, that the C-terminus of Galpha is mainly responsible for bringing the G protein into the close proximity of the receptor's intracellular domains, thus ensuring efficient coupling, rather than having a particular role in transmitting the signal. To confirm this conclusion, we show that two proteins interacting with each other (such as Snf1 and Snf4, or Ras and Raf), each of them fused either to the receptor or to the chimeric Galpha, allow efficient signal transduction.

Four RNA motifs are known that catalyse site-specific cleavage in the presence of Mg2+ ions, all discovered in natural RNAs. In a single in vitro selection experiment we have isolated representatives of five novel classes of Mg(2+)-dependent ribozymes. Small versions of three of these showed that a very simple internal loop type of secondary structure is responsible for the activity. One of these was synthesized in a bimolecular form, and compared directly with the hammerhead ribozyme; for the new ribozyme, the cleavage step of the reaction is much faster than the spontaneous rate of phosphodiester bond cleavage, yet substantially slower than that for the hammerhead. The results suggest that many more Mg(2+)-dependent self-cleaving RNA sequences can be found.

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.

We have purified to apparent homogeneity a type II DNA topoisomerase from Xenopus laevis oocyte nuclei (germinal vesicles, or GV). The most pure preparations contain a single polypeptide of 175,000 daltons as determined by SDS-gel electrophoresis. The enzyme changes the linking number of DNA circles in steps of two and reversibly knots or catenates DNA rings. No gyrase activity is detectable and ATP is required.

We have produced a highly purified preparation of the Xenopus laevis splicing endonuclease (XlaI RNase). The purified enzyme correctly cleaves tRNA precursors, creating substrates for subsequent ligation. The 5'-half molecules have a 2',3' cyclic phosphate at their 3' termini. Assuming that splicing enzymes recognize primarily structural elements in the 'mature domain', we have been studying the conformation of three splicing-defective precursors made from mutants of the yeast tRNALeu3 gene. The mutations alter base-pairing in the D-stem region and two of the mutants are absolute defectives. Enzymatic probing of the structures of the altered tRNA precursors shows that the structural perturbations in these mutants are localized on the 'inside' of the 'L'-shaped three-dimensional structure. The implications of this finding for the recognition process are discussed.