Mol Gen Genet (1988) 212:382-385 © Springer-Verlag 1988 An unlinked 5 S ribosomal RNA gene in the archaebacterium, Thermococcus celer Doreen E. Culham and Ross N. Nazar Department of Molecular Biology and Genetics, University of Guelph, Gueph, Ontario, Canada N1G 2Wt Summary. We have determined the nucleotide sequence of an unlinked 5 S rRNA gene region from a thermophilic archaebacterium, Thermococcus celer. This 5 S rRNA gene is flanked by a single tRNA Asp sequence and appears to be transcribed as part of a very short operon consisting of only two gene sequences. Comparative studies indicate features in the 5' and 3' flanking sequences, which bear similarity with promoter and termination signals in eubac- teria, but also reflect unusual features found in at least some archaebacteria. The evolution of this unlinked operon and the unusual features are discussed. Key words: Thermococcus celer - Unlinked 5 S rDNA Nucleotide sequence In all organisms, the mature large ribosomal RNA compo- nents (16-18 and 23~8 S rRNAs) are cleaved from a com- mon precursor molecule. In eubacteria, the 5 S RNA se- quence is also transcribed as part of this common precursor (see Nomura and Post 1980) but the 5 S RNA of the eukar- yotic cytoplasmic ribosome is transcribed from a separate gene by an alternative RNA polymerase III (see Nazar 1982). Although not co-transcribed, in lower eukaryotes such as Saccharomyces, the 5 S genes remain linked to the rDNA as part of the same repeating DNA unit; in higher eukaryotes this association is entirely absent. Because these evolutionary changes are both substantial and intriguing they are the subject of considerable speculation. Since alter- native 5 S genes are activated during different states of de- velopment in at least some eukaryotes (see Brown and Schlissel 1985) the possibility exists that some of these evo- lutionary changes may be directed towards cellular regula- tion. Recent studies (Jarsch et al. 1983; Neumann et al. 1983; Wich et al. 1984) on the organization of ribosomal RNA genes in archaebacteria have revealed at least three organ- isms; Methanococcus, Sulfolobus and Thermococcus, with extra, apparently unlinked, 5 S rRNA genes. As these could represent early examples in the evolution of unlinked 5 S RNA genes further study has been undertaken. In two strains of Methanococcus, DNA cloning and sequencing (Wich et al. 1984, 1987) has shown that the unlinked gene is localized in a cluster of tRNA gene sequences and may Offprint requests to. R.N. Nazar E H X U S X P P C SX X C X P T AA H C T S E T Y V ¥ T Y ~r ¥ Y YT ¥ ¥ ¥ Y Y VY ¥ ¥ y y Y I ID ii i D 11 I , ~ : I It 0 2 0.4 0 0.SKb Fig. 1. Determination of the nucleotide sequence for an unlinked 5 S rRNA gene region from Thermococcus celer. A 2-Kb EcoRI (E) fragment containing the unlinked 5 S rRNA gene was isolated by plaque hybridization from a 2 Charon 3A genomic library, and subcloned using the pBR322 cloning vector. The nucleotide sequence for a 1,055-base portion of this fragment was determined by further cleaving the EeoRI fragment with restriction endonucle- ase CfoI (C), HinfI (H), HaeIII (A), HpaII (P), SstI (S), StuI (U), TaqI (T), or XhoI (X) and degrading it chemically by the sequenc- ing techniques of Maxam and Gilbert (1980). The arrows indicate the direction and extent of the sequences that were obtained be expressed as part of a separate operon. In this study an unlinked gene in a second genus of archaebacterium, Thermococcus celer, has been isolated and characterized. Sequence analyses indicate that this 5 S RNA gene is, ap- parently, part of a much smaller operon and may be co- transcribed with a single tRNA Asp sequence. An earlier study on the 5 S rRNA genes of T. celer (Neumann et al. 1983) suggested that in this organism one type of 5 S DNA sequence was linked with the 16 and 23 S rDNAs in a very large (> 20 kb) genomic EcoRI-diges- tion fragment while a second, unlinked copy, was present in a much shorter 2-kb fragment. To clone this shorter fragment specifically, DNA was extracted (Cryer et al. 1975) from Thermococcus celer cells, strain Vu13, DMS 2476 (generously provided by W. Zillig) and a ~ Charon 3A vector (Blattner et al. 1977) was used to construct a genomic library, thereby excluding the much longer EcoRI- digestion fragment. DNA from a complementary plaque (2Ch 3A-TC2000) was digested with EcoRI endonuclease and further subcloned into pBR322; colony hybridization was used to select complementary clones (see Maniatis et al. 1982). As indicated in Fig. 1, subsequent cleavages with restriction enzymes and hybridization analyses (Bittner et al. 1980; Seed 1982) were used to identify complementary fragments and prepare them for sequence determination (Maxam and Gilbert 1980). About 1 kb of sequence was determined (Fig. 2) including the 5 S rRNA sequence and