Conservation of substrate recognition mechanisms by tRNA splicing endonucleases.

Accuracy in transfer RNA (tRNA) splicing is essential for the formation of functional tRNAs, and hence for gene expression, in both Eukaryotes and Archaea. The specificity for recognition of the tRNA precursor (pre-tRNA) resides in the endonuclease, which removes the intron by making two independent endonucleolytic cleavages. Although the eukaryal and archaeal enzymes appear to use different features of pre-tRNAs to determine the sites of cleavage, analysis of hybrid pre-tRNA substrates containing eukaryal and archaeal sequences, described here, reveals that the eukaryal enzyme retains the ability to use the archaeal recognition signals. This result indicates that there may be a common ancestral mechanism for recognition of pre-tRNA by proteins.

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.

Cleavage site recognition by the tRNA splicing endoribonuclease.

A single tRNA-splicing endoribonuclease can cleave several precursors. In addition to the conserved nucleotides (nt), there are positions in the mature domain that, though not always occupied by the same nt, nevertheless play a fundamental role in intron excision reaction. The elements of the recognition set (invariant nt, nt at the cardinal positions) can contribute to the overall recognition process by providing either a direct contact for the enzyme or by dictating the spatial orientation of nt that are directly contacted.

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.

Participation of the intron in the reaction catalyzed by the Xenopus tRNA splicing endonuclease.

Introns have generally been assumed to be passive in the transfer RNA splicing reaction. Experiments have now been done showing that the endonuclease is able to cut a precursor provided that a base in the single-stranded loop of the intron can pair with the base of the 5' exon situated at the position that immediately follows the anticodon stem (position 33 in the yeast tRNA isoacceptor pre-tRNA(Leu)3, position 32 in yeast pre-tRNA(Phe)). The elucidation of the role of the intron reveals that in addition to the conserved bases, there are positions in the mature domain which, although not necessarily occupied by the same base in all pre-tRNA's, nevertheless have a fundamental role in the splicing reaction. These positions are termed cardinal positions.

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.

Binding and cleavage of pre-tRNA by the Xenopus splicing endonuclease: two separable steps of the intron excision reaction.

We have constructed three base-substitution mutants of the yeast tRNALeu3 gene. In two of them the ability to form an extended anticodon stem is lost. In the first mutant the bases encoding the anticodon change from TTG to GAC (positions 37, 36, 35); in the second, the nucleotides encoding the region of the intron that base-pair with the anticodon change from CAA to GTC (positions 48, 47, 46). The third is a double mutant characterized by both substitutions described above so that its ability to form an extended anticodon stem is restored. The precursors derived from the two single mutants are accurately spliced in the X. laevis germinal vesicles (GV) extract: pairing of the anticodon with the intron, therefore, is not required for the splicing reaction. The precursor derived from the double mutant is not spliced, indicating that the new extended anticodon stem exerts an inhibitory action. Since the double mutant precursor binds to the purified splicing endonuclease, binding and cleavage occur as two separable steps in the intron excision reaction.

Role of RNA structure in splicing: excision of the intervening sequence in yeast tRNA3leu is dependent on the formation of a D stem.

A substitution mutant of the yeast tRNA3leu gene results in the sequence change of GCC to AAA at positions 10, 11, and 12 in the noncoding strand. The ability to form a D stem is lost. Transcription in the heterologous Xenopus germinal vesicle system is not drastically affected, but splicing of the tRNA precursor does not occur. To determine whether this effect is caused by the change in sequence or the change in conformation we constructed two new mutants. In one, mutation results in the sequence change of GGC to TTT at positions 24, 25, and 26. The ability to form a D stem is lost; transcription is unaffected, but excision of the intron does not occur. The other, a double mutant, is characterized by both substitutions described above, and the ability to form a D stem is retained. The precursor derived from the double mutant is accurately spliced in X. laevis germinal vesicle extracts, therefore excision of the intervening sequence appears to depend on the formation of a D stem.

Mutation in the a block of the yeast tRNAleu3 gene that allows transcription but abolishes splicing and 5'-end maturation.

A three-base substitution mutant of the yeast tRNALeu3 has been constructed. The mutation, introduced through the use of a heptadecanucleotide as a site-specific mutagen, is localized in the anterior portion of the promoter and results in the inability to form a D stem. The mutant is active in transcription, but maturation of the 5' terminus and splicing are abolished. The results are discussed in the light of a recently proposed model for initiation of transcription of eucaryotic tRNA genes.

Purification and characterization of Xenopus laevis topoisomerase II.

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.

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.

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.

DNA supercoiling by Xenopus laevis oocyte extracts: requirement for a nuclear factor.

A purified system is described for the introduction of negative supercoils into simian virus 40 DNA. The system consists of histones H2A, H2B, H3, and H4, DNA-relaxing enzyme, and a purified factor from Xenopus laevis stage 6 oocyte nuclei. The nuclei are prepared en masse by the technique of F. Scalenghe, M. Buscaglia, C. Steinheil, and M. Crippa [(1978) Chromosoma 60, 299-308]. The supercoiled simian virus 40 DNA prepared by this method is indistinguishable from simian virus 40 supercoiled DNA prepared from infected monkey cells.

DNA-relaxing activity and endonuclease activity in Xenopus laevis oocytes.

Complex simian virus 40 DNA produced by a soluble cell-free extract derived from stage 6 oocytes of Xenopus laevis consists of fully relaxed circles (i.e., with no superhelical turns). An endonuclease and a DNA-relaxing protein, either or both of which could be responsible for the relaxation of the complex DNA, have been purified from the extract. The endonuclease(s) produces nicked circles (having a single-strand scission) and linear full-size molecules. The DNA-relaxing protein is in the nucleus, has a molecular weight of apporximately 70,000, and is able to remove both negative and positive superhelical turns.

Effect of Xenopus laevis oocyte extract on supercoiled simian virus 40 DNA: formation of complex DNA.

A soluble cell-free extract derived from stage 6 Xenopus laevis oocytes is described. From supercoiled simian virus 40 DNA the extract produces nicked circles (having a single-strand scission), linear molecules of full unit size, shorter length fragments, and various forms of complex DNA.