Examples of splicing motifs present in archaeal pre-tRNAs bearing introns at positions 37/38 and others. (A,B,C) The cloverleaf sequences of three characteristic tDNAs harboring an intron between nt 37 and 38. (D,E) Introns located at unconventional positions. Small arrows show the intron splicing position, always located two bases downstream of the H1 and H2 strands of the central H helix. The central 4-bp H helix is highlighted with a gray background. (A) As examplified with pre-tRNA-Lys (UUU) from S. tokodaii, this panel shows the most often encountered splicing motif hBHBh′ (67 times over a total of 103 cases; see Supplementary Material). In this case, the exonic h helix (boxed) is formed by the antiparallel strands h1 and h2 of the anticodon stem. The central H helix (gray background) includes the first two bases of the anticodon (boxed). The third helix, h′ (boxed), is formed within the intron (here, 25 nt in length). The three h, H, and h′ helices allow for the formation of two bulges, B1 and B2. The intron is always spliced symmetrically two bases downstream of strands H1 and H2 of helix H. (B) In the pre-tRNA-Ser (UGA) of S. tokodaii, the intron (11 nt) located between nt 37 and 38 is too short to allow the helix h′ and the bulge B1 to be formed. This type of relaxed splicing motif is referred to as relaxed hBH. (C) In the pre-tRNA-Thr (UGU) from S. tokodaii, although this pre-tRNA harbors a 17-nt-long intron, the intron–exon junction can only fold into a relaxed hBH type of splicing motif. No complementary bases are present within the intron, preventing the formation of the intronic h′ helix. (D) Hypothetical folding of a 20-nt intron located at position 29/30 within the pre-tRNA-Val (GAC) of P. aerophilum. Bases 20–34 are replaced within the conventional cloverleaf scheme by asterisks and are drawn in an alternative 2D structure that fits the splicing motif requirement. In this pre-tRNA, formation of the canonical splicing motif hBHBh′ obviously competes with the formation of the regular D- and anticodon stems. (E) Hypothetical folding of a 36-nt intron located at position 45/46 in the pre-tRNA-Lys (CUU) of A. pernix. Here, bases 35–51 of the conventional cloverleaf are replaced by asterisks and are redrawn to fit a relaxed splicing motif HBh′, in which a helix h cannot be formed because of the absence of the needed complementary bases (dotted box). This putative folding obviously competes with the formation of the regular anticodon and T-stems.
