ABSTRACT
The splicing endonuclease is responsible for recognition and excision of tRNA introns. Introns
are predominandy found in the acceptor stem and are thought to minimally interrupt the mature tRNA structure that comprises the acceptor stem, the D stem and the T'PC stem.63*65 In eukarya, the intron is located invariably at the same position between positions 37 and 38 in the anticodon stem. While archaeal introns are often located at this same position in the anticodon stem, there are other locations where introns might be found.66 Early work done in the Abelson and Tocchini-Valentini laboratories determined the recognition specificity of the eukaryotic splicing endonucleases. The location of the nuclear tRNA introns relative to the mature domain is conserved and is one of the key elements necessary for recognition of the intron-exon boundary.2,67'69 Two nucleotides are also necessary for recognition. The cardinal position 1 (CPI) is located two bases upstream of the anti codon and base pairs with an intron nucleotide three bases upstream of the 3' splice site, forming an anticodon-intron (A-I) pair. The cardinal position 2 (CP2) is located one base downstream of the 3' splice site.2,70,71 Studies on the yeast splicing endonuclease demonstrated how these key elements are utilized for substrate recognition using a “ruler mechanism”. The enzyme measures down five base pairs from the mature tRNA domain to locate the 5' splice site and uses the A-I pair to correctly locate the 3' splice site.69,71 Archaeal splicing endonucleases differ from their eukaryotic counterparts in that they do not require the mature tRNA domain to locate the splice sites; rather, these endonu cleases rely on an RNA secondary structure motif termed the bulge-helix-bulge (BHB) motif that is necessary for cleavage of most archaeal splice sites.72 This motif is generally comprised of a pair of three-nucleotide bulges separated by a four base-pair helix, though variations in this motif have been identified for some archaeal organisms.66 While there are significant differences in the recognition of tRNA substrates between archaea and eukarya, some similarities in the local structural features around the splice sites are observed. The archaeal BHB motif contains an A-I pair at the 3' splice site, as well as at its 5' splice site due to the pseudosymmetric nature of the motif.2,71 Furthermore, the distance between the splice sites is similar in both archaeal and eukaryotic substrates.
