STRUCTURAL FEATURE OF THE INITIATOR TRANSFER-RNA GENE FROM PYRODICTIUM-OCCULTUM AND THE THERMAL-STABILITY OF ITS GENE-PRODUCT, TRNA(I)(MET)

Pyrodictium occultum is a hyperthermophilic archaeum that grows optima lly at 105 degrees C. To study how tRNA molecules in P occultum are th ermally stabilized, we isolated the initiator tRNA gene from the organ ism using a synthetic DNA probe of 74 bp containing the known nucleoti de sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence rev ealed that the Pyrodictium initiator tRNA gene has no introns, and tha t the 3' CCA terminus is encoded. The tRNA gene also contained a uniqu e TATA-like sequence, AAGCTTATAA, which is likely the promoter propose d for archaeal tRNA genes, -50 bp upstream of the 5' end of the tRNA c oding region. In the region adjacent to the 3' end of the tRNA coding region, there was a six G-C base pair inverted repeat followed by a C- rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A3 7, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (T-m) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 d egrees C, which is only two degrees lower than that calculated from th e G-C content in the stem regions of the tRNA. In contrast, the T-m of the natural initiator tRNA isolated from P occultum was over 100 degr ees C. Analysis of digests of purified Pyrodictium initiator tRNA by m eans of HPLC-mass spectrometry and [P-32] post-labeling, indicated tha t the tRNA contains a variety of modified nucleosides. These results s uggest that the extraordinarily high melting temperature of P occultum tRNA(i)(Met) is due to posttranscriptional modification.