E Mattoccia

In vitro catenation and decatenation of DNA and a novel eucaryotic ATP-dependent topoisomerase

Extracts from X. laevis germinal vesicles interlock duplex DNA circles to form catenanes. The catenation activity requires Mg++ and ATP. Negatively supercoiled or relaxed DNA can be used as substrates for the catenation reaction. Homology between donor and acceptor DNA is not required, since catenanes are formed between DNA molecules with unrelated sequences. In the course of the isolation of the activity responsible for the catenation reaction, we discovered a new ATP-dependent topoisomerase. The fractions containing the novel topoisomerase catenate and decatenate DNA, the ionic strength dictating which of the two opposing reactions will occur.

The Eucaryal tRNA Splicing Endonuclease Recognizes a Tripartite Set of RNA Elements

The tRNA splicing endonuclease cleaves intron-containing tRNA precursors on both sides of the intron. The prevailing belief has been that the enzyme binds only to the mature domain through the invariant bases. We show instead that, for recognition, the endonuclease utilizes distinct sets of structural elements, several of which are within the intron. One subset of recognition elements, localized in the mature domain, is needed for recognition of both cleavage sites, while two other subsets, localized at the exon-intron boundaries, are used for recognition of either one or the other cleavage site. The two cleavage sites are essentially independent: neither is required by the other for cleavage to take place. These results support a two-active-site model for the eucaryal endonuclease.

Site selection by the tRNA splicing endonuclease of Xenopus laevis

To investigate the mechanism by which the purified Xenopus tRNA splicing endonuclease recognizes its splice sites, we utilized yeast pre-tRNA(3Leu) and pre-tRNA(Phe) variants constructed by in vitro mutagenesis. We found that the endonuclease interacts with conserved features of the mature tRNA domain. In particular, U8 and C56 may be examples of contact points between protein and RNA. Given that there are no conserved sequences at the splice junctions, the specificity of cutting at both splice sites is determined by the length of the anticodon stem. Although in general, the sequence of the intron is unimportant for splicing, there are some structural requirements.

Separation of RNA transcription and processing activities from X. laevis germinal vesicles

A procedure suitable for en masse preparation of germinal vesicles (GV) from X.laevis oocytes (Scalenghe et al., 1978) has been adapted for studies of transcription. Extracts from GV contain activities for transcription of tRNA genes and for processing the transcription product. The two activities have been separated by column chromatography. One fraction allows synthesis of tRNA precursor molecules in the presence of X.laevis RNA polymerase III. Another fraction contains the activity that cuts and splices those precursors which contain an intervening sequence. Transcription occurs faithfully on linear DNA fragments.