Transfer RNA intron processing in the halophilic archaebacteria

Abstract

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.

Identification of BHB splicing motifs in intron-containing tRNAs from 18 archaea: evolutionary implications

“…They identified an unusually located 18-nt-long intron in the variable loop of the precursor tRNAGly (CCC) of the extreme thermophile Thermophilum pendens (Kjems et al 1989b). In this pre-tRNA, the exonintron boundaries were able to fold into a structure similar but not identical to the characteristic bulge-helix-bulge (BHB) usually found in the intron-exon boundaries located at position 37/38 of pre-tRNAs (Kaine et al 1983;Daniels et al 1985;Daniels and Dougas 1986;Kaine 1987;Datta et al 1989; Thompson et al 1989; Thompson and Daniels 1990;Kleman-Leyer et al 1997) and in the long Watson-Crick base-paired stems of a few rRNA precursors of hyperthermophilic Archaea (Kjems and Garrett 1988;Dalgaard and Garrett 1992;Burggraf et al 1993;Durovic and Dennis 1994;Lykke-Andersen and Garrett 1994;Dennis et al 1998;Itoh et al 1998;Nomura et al 1998;Ciammaruconi and Londei 2001). More recently, introns were discovered in the anticodon loop between nt 32 and 33 in the pre-tRNA-Pro (GGG) of Methanobacterium thermoautotrophicum (Smith et al 1997), in the D-loop and anticodon stem of several tDNAs of Sulfolobus solfataricus (She et al 2001), and in many other locations in tDNAs of Pyrobaculum aerophilum (Fitz-Gibbon et al 2002).…”

In silico screening of archaeal tRNA-encoding genes having multiple introns with bulge-helix-bulge splicing motifs

“…All of these tDNAs have a bulge-helix-bulge (BHB) structural consensus, which is also formed in most archaeal pre-rRNA and pre-mRNA introns (Tang et al 2002;Watanabe et al 2002;Yoshinari et al 2006) around their exon-intron boundaries (Kaine et al 1983;Daniels et al 1985;Datta et al 1989;Thompson et al 1989;KlemanLeyer et al 1997). The canonical BHB is a single-hairpin structure that consists of two bulges (B) of 3 nucleotides (nt) separated by a central helix (H) of 4 base pairs (bp) within consensus motif sequences (hBHBh9).…”

Two reactions of Haloferax volcanii RNA splicing enzymes: Joining of exons and circularization of introns

“…1A,D, 5A), formed by pairing of the regions near the two intron-exon junctions. The splicing endonucleases recognize these BHB structures and cleave each of the two strands between the middle and 3Ј residues of the three-nucleotide bulges that are separated by four base pairs (Kjems et al 1989a; Thompson et al 1989; Thompson and Daniels 1990;Lykke-Andersen andGarrett 1994, 1997). Archaeal splicing endonucleases are also involved in rRNA processing, cleaving within the BHB structures present in the processing helices formed by pairing of the 5Ј and 3Ј flanking regions of the rRNAs (Durovic and Dennis 1994;Dennis et al 1998;Ciammaruconi and Londei 2001;Tang et al 2002).…”