Expression of a yeast intron-containing tRNA in the archaeon Haloferax volcanii.

Expression of the yeast tRNAPro(UGG) gene in Haloferax volcanii resulted in the production of a single stable transcript that had not undergone intron processing or processing of 5' and 3' flanking sequences. Mutation of the exon-intron boundary region of this RNA to produce a precursor RNA with the preferred halobacterial consensus exon-intron boundary structure did not restore intron processing. Processing of 5' and 3' flanking sequences was restored when the acceptor stem U6-U67 pair was changed to A6-U67. The significance of these results in defining the recognition requirements of tRNA maturation enzymes in the halophilic domain Archaea is discussed.

In vivo processing of an intron-containing archael tRNA.

In vitro studies on the processing of halobacterial tRNA introns have led to the proposal that archaeal and eukaryotic tRNA intron endonucleases have distinctly different requirements for the recognition of pre-tRNAs. Using a Haloferax volcanii in vivo expression vector we have examined the in vivo processing of modified forms of the halobacterial intron-containing tRNA(Trp) gene. As observed in vitro, changes in the exon-intron boundary structure of this pre-tRNA block processing. Intron sequences, other than those at the exon-intron boundaries, are not essential for processing in vivo. We also show that conversion of the tryptophan anticodon to an opal suppressor anticodon is tolerated when the exon-intron boundary structure is maintained.

The RNA component of RNase P from the archaebacterium Haloferax volcanii.

RNase P, an endoribonuclease responsible for generating the mature 5' termini of tRNA precursors, is composed of both RNA and protein. It has been demonstrated that the eubacterial RNase P RNA will, under the appropriate reaction conditions, exhibit catalytic activity in vitro. Evidence has not been obtained for catalytic activity by the RNAs of eukaryotic RNase P enzymes. Using a cDNA probe prepared from RNA copurifying with RNase P activity from the archaebacterium Haloferax volcanii, we have characterized the gene encoding the RNase P RNA. The proposed transcript from this gene can assume a structure resembling the eubacterial RNase P RNA and includes many of the highly conserved sequences of these RNAs. This RNA was incapable of cleaving pre-tRNA substrates in the absence of protein under a variety of in vitro conditions. Catalytic activity was observed when this RNA was combined with the protein subunit of the Bacillus subtilis RNase P complex. Similarities among the archaebacterial, eubacterial, and eukaryotic RNase P RNA sequences and structures are discussed.

An expression vector for the archaebacterium Haloferax volcanii.

The recent development of an efficient transformation method and shuttle vectors for Haloferax volcanii has set the stage for rapid progress in archaebacterial molecular biology. We describe a shuttle-expression vector that can be selected for and maintained in either H. volcanii or Escherichia coli and permits the expression of cloned genes in H. volcanii. The vector, pWL204, was constructed by incorporating an H. volcanii tRNA(Lys) gene promoter into a derivative of the H. volcanii-E. coli shuttle vector pWL102. The vector has been used to express a modified, intron-containing, H. mediterranei tRNA(Trp) gene (tRNA(Trp)-O167). Transcription from the tRNA(Lys) gene promoter in vivo was detected by Northern (RNA) analysis with an oligonucleotide probe complementary to the unique intron sequence of tRNA(Trp)-O167. Dependence of transcription on the tRNA(Lys) promoter was demonstrated by the absence of transcription when the promoter sequence was deleted from the vector and by mapping the transcription initiation site by primer extension.

Transformation of a conditionally peptidoglycan-deficient mutant of Staphylococcus aureus with plasmid DNA.

A simple and reliable method for polyethylene glycol-induced plasmid transformation of a temperature-sensitive peptidoglycan-deficient mutant of Staphylococcus aureus is described. The procedure uses strains carrying the tofA372 mutation grown under conditions that yield osmotically fragile cells capable of efficient wall regeneration. The peptidoglycan-deficient cells were transformed with plasmids pE194 and pI258 at frequencies comparable with those obtained with protoplasts prepared with lysostaphin treatment. A readily portable tofA372 mutation was constructed by isolating an insertion of the erythromycin resistance transposon Tn551 adjacent to tofA372. tofA372 was shown by protoplast fusion and transformation analyses to be in the gene order hly-421-omega [Chr::Tn551]1059-tofA372-uraB232-omega [Chr::Tn916]1101-thrB106 on the chromosome of S. aureus NCTC 8325.

Transfer RNA intron processing in the halophilic archaebacteria.

An in vitro assay system has been developed for the Halobacterium volcanii tRNA intron endonuclease using in vitro generated precursor RNAs. A partially purified enzyme preparation is capable of precise and accurate excision of the intron from the halobacterial tRNA(Trp) precursor. The cleavage reaction produces products having 5' hydroxyl and 2',3' cyclic phosphate termini. Processing of precursor molecules containing deletions within the exon regions indicates that the halobacterial endonuclease does not require intact mature tRNA structure in the substrate; this is in contrast to the eukaryotic endonuclease enzyme that has an absolute requirement for these structures. The large halobacterial tRNA(Trp) intron does not appear to be a primary site for recognition by the endonuclease, however, its removal affects cleavage efficiency. Through a comparison of the structural and sequence features of the halobacterial substrates and the precursors of other archaebacterial intron-containing precursors, a common element is proposed for the recognition of substrates by intron endonuclease.