tRNA processing in the yeast, Saccharomyces cerevisiae, as well as in other eukaryotes, often involves a splicing step. The substrates for tRNA splicing are a subset of tRNA precursors which contain introns ((1, 2) see Ref. 3). Unlike the case of introns in mRNA precursors, the introns in precursor tRNAs do not have obvious conserved sequences at their splice junctions or within the intron; the major common feature these introns share is their location, which is one base removed from the 3' -end of the anticodon (4). In contrast to the splicing of mRNA precursors, which involves several different ribonucleoproteins in complexes (for a review, see Ref. 5), the removal of introns from yeast tRNA precursors has been shown to be a relatively simple process involving two enzymes (6) (Fig. 1).
After an endonuclease excises the intron to leave 5' -hydroxyl and 2' ,3' -cyclic phosphodiester ends, tRNA ligase joins the cognate 5' and 3' tRNA half-molecules (7, 8).
tRNA ligase protein is a single 90-kDa polypeptide which is likely to contain three activities required for the tRNA splicing reaction (9). tRNA ligase phosphorylates the 5' terminus of the 3' half-tRNA in the presence of ATP; opens the 2' ,3' -cyclic phosphodiester bond of the 5' half-tRNA, leaving a 2' -phosphomonoester; and, in a second ATP- dependent reaction, ligates the two tRNA halves (7) (see Fig. 1 ). Ligation occurs through an adenylylated enzyme intermediate, presumably at a lysine residue as it is in T4 RNA ligase (7, 10). The AMP moiety of this intermediate is transferred to the 5' -phosphate of . the 3' half-tRNA to form a 5' ,5' -phosphoanhydride bond and is then released upon ligation of the two tRNA halves (9). tRNA ligase has been purified to near homogeneity, and the three activities described above cosediment with this protein in the final glycerol gradient step (9). (Another enzyme presumably removes the 2' -phosphate which remains at the splice junction after the action of tRNA ligase.)
In order to study the functional domains of the tRNA ligase protein, we have cloned the gene from S. cerevisiae (9). We present here its DNA sequence, which encodes a . protein of 827 amino acids (95.4 kDa). The ligase amino acid sequence does not resemble
that of other proteins with similar activities.
The transcript for tRNA ligase starts at two major sites, 104 and 125 nucleotides upstream from the initiation codon. Near the 5' -end of tRNA ligase, there are two other open reading frames, ORF1 1 and ORF2. ORF1 begins upstream from tRNA ligase and overlaps the beginning of the ligase gene. We discuss experiments suggesting that its translation is not essential. The ORF2 reading frame begins 342 nucleotides upstream from the ligase initiation codon and proceeds in the opposite direction. This gene is transcribed to produce a 2.1-kilobase RNA that begins 125 nucleotides upstream from the tRNA ligase transcript.
EXPERIMENTAL PROCEDURES
Strains and Plasmids. Escherichia coli JMlOl (11) was used for preparing M13 phage for sequencing and restriction analysis. Yeast strain TSY6-11 Ba (MAT a prcl -6.2 pep4-3 ura3 -52 leu2-3, 112 his4) was used as the host for analysis of transcription products and transcript mapping and was obtained from Dr. S. Emr (California Institute of Technology). Yeast strain SWY497 (our nomenclature) has an ochre mutation within ORFl constructed by site-directed oligonucleotide mutagenesis of single-stranded Ml3 phage (12, 13) followed by standard yeast gene replacements methods (14, 15) using yiPS (15) as the integrating vector. EMPY20 is the parent yeast diploid for SWY497 and was constructed by mating SS328 (MATa his36.200 lys2-801a ade2-101" uraJ-52 GAL suc2) to SS330 (MATa his36.200 tyr1 ade2-JOJ" uraJ-52 GAL suc2). SS328 and SS330 were obtained from Dr. S. Scherer (University of Minnesota): Construction of plasmid pUC12- RLG was described previously (9). Plasmid pEP99 (our nomenclature) is a derivative of yEP24 which contains the entire tRNA ligase gene as well as 5 kilobases of upstream DNA. This plasmid was obtained by screening an E. coli library of yeast genomic DNA (16) for inserts that hybridize to the ligase DNA (the library was provided by Dr. J.
Campbell). Screening was performed as described by Maniatis et al. (17). Transformation of this plasmid and its parent, yEP24 (18), into strain TSY6-11Ba was done as described byitoetal. (19).
M13 Template Construction and Sequencing. Four unique restriction sites were used to subclone the 4-kilobase EcoRI insert of plasmid pUC12-RLG into phage M13 for sequencing (see Table I). Phage were purified by CsCl density gradient centrifugation prior to sequencing (20). These templates were sequenced by the Sanger dideoxy sequencing method (21) by first using the Ml3 sequencing primer and then by priming with synthetic oligonucleotides as needed (22, 23). These oligonucleotide primers were
synthesized on an Applied Biosystems 380A DNA synthesizer (deoxyoligonucleotides were provided by Dr. S. H. Horvath, California Institute of Technology).
Preparation of RNA. Yeast cells were grown in SM minimal minus uracil media (24)
to 2 X 107 cells/mi. After harvest, RNA was extracted by disruption of the cells with glass beads in the presence of cold phenol (25). The polyadenylated RNA was separated from total RNA on oligo(dT)-cellulose (17).
RNA Analysis. 3 f.lg of polyadenylated RNA was resolved on a 1.5% formaldehyde-
agarose gel and transferred to nitrocellulose (17). Single-stranded DNA probes used in the Northern blot were made by primer extension with [a-32P]dCTP across the ligase insert of the Ml3 template followed by restriction cutting, denaturation of the duplex DNA, and isolation of the labeled single-stranded DNA. The primers and templates were selected from those listed in Table I and Fig. 2. Prehybridization, hybridization, and washing of the Northern blot were carried out according to Thomas (26).
Transcript mapping by primer extension using 20-40 units of reverse transcriptase was carried out as described previously (27) with 3 f.lg of polyadenylated RNA. S 1 nuclease analysis was performed as described (28, 29) with 3 f.lg of polyadenylated RNA and the appropriate kinased and primer-extended oligonucleotides. The oligonucleotide used as a primer for detecting the tRNA ligase transcript is complementary to the first 30 nucleotides of the tRNA ligase coding region.
Sequence Analysis. Sequence data was assembled, translated, and analyzed using
Versions 4.2 and 4.5 of the Intelligenetics GENED, GEL, SEQ, and PEP programs of the BIONET™ National Computer Resource for Molecular Biology. All protein sequences used in homology searches were obtained using the Intelligenetics QUEST, !FIND, and XFASTP programs with the National Institutes of Health sequence and National
Biomedical Research Foundation protein data bases in BIONET™, unless otherwise referenced.
Materials. M13 sequence primer was from New England Biolabs. [a-32P]dNTPs were
purchased from Amersham Corp. dNTPs, dideoxy-NTPs, and S I nuclease were from Pharmacia LKB Biotechnology Inc. Klenow fragment of DNA polymerase was purchased from Bethesda Research Laboratories. Reverse transcriptase was from Boehringer Mannheim.