Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105°C. To study how tRNA molecules in P occultum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide 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 revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal tRNA genes, -50 bp upstream of the 5' end of the tRNA coding 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, A37, 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°C, which is only two degrees lower than that calculated from the 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°C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32p] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(i)(Met) is due to posttranscriptional modification.
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