Experimental confirmation of a whole set of tRNA molecules in two archaeal species

Abstract

Based on the genomic sequences for most archaeal species, only one tRNA gene (isodecoder) is predicted for each triplet codon. This observation promotes analysis of a whole set of tRNA molecules and actual splicing patterns of interrupted tRNA in one organism. The entire genomic sequences of two Creanarchaeota, Aeropyrum pernix and Sulfolobus tokodaii, were determined approximately 15 years ago. In these genome datasets, 47 and 46 tRNA genes were detected, respectively. Among them, 14 and 24 genes, respectively, were predicted to be interrupted tRNA genes. To confirm the actual transcription of these predicted tRNA genes and identify the actual splicing patterns of the predicted interrupted tRNA molecules, RNA samples were prepared from each archaeal species and used to synthesize cDNA molecules with tRNA sequence-specific primers. Comparison of the nucleotide sequences of cDNA clones representing unspliced and spliced forms of interrupted tRNA molecules indicated that some introns were located at positions other than one base 3' from anticodon region and that bulge-helix-bulge structures were detected around the actual splicing sites in each interrupted tRNA molecule. Whole-set analyses of tRNA molecules revealed that the archaeal tRNA splicing mechanism may be essential for efficient splicing of all tRNAs produced from interrupted tRNA genes in these archaea.

Figure 1

Overall strategy used to confirm predicted tRNA molecules in A. pernix K1.

Figure 2

Agarose electrophoresis patterns of RT-PCR-amplified fragments. (A) Amplified without RT (reverse transcriptase); (B) Amplified with RT. Lanes 1 to 13 contain fragment that were amplified with primer sets 15–27 summarized in previous work; these primers were described previously [9]. M: 100 bp ladder marker.

Figure 3

Summary of the tRNA introns identified in A. pernix K1. Each label includes the tRNA species, the respective intron positions from the 5' end of the respective tRNA, and the lengths of the introns. Characters within parentheses indicate the nucleotide one base 5' from the intron border.

Figure 4

Proposed structures of the unspliced forms of interrupted tRNA molecules in A. pernix K1. Splicing sites and anticodon regions are indicated by short arrows and boxes, respectively. (A) tRNA^Arg(UCU); (B) tRNA^Asp(GUC); (C) tRNA^Met(CAU-1); (D) tRNA^Met(CAU-2); (E) tRNA^Thr(CGU); (F) tRNA^Pro(GGG); (G) tRNA^Cys(GCA); (H) tRNA^Tyr(GUA); (I) tRNA^Thr(UGU-1); (J) tRNA^Trp(CCA); (K) tRNA^Lys(CUU); (L) tRNA^Lys(UUU); (M) tRNA^Pro(CGG).

Figure 4

Proposed structures of the unspliced forms of interrupted tRNA molecules in A. pernix K1. Splicing sites and anticodon regions are indicated by short arrows and boxes, respectively. (A) tRNA^Arg(UCU); (B) tRNA^Asp(GUC); (C) tRNA^Met(CAU-1); (D) tRNA^Met(CAU-2); (E) tRNA^Thr(CGU); (F) tRNA^Pro(GGG); (G) tRNA^Cys(GCA); (H) tRNA^Tyr(GUA); (I) tRNA^Thr(UGU-1); (J) tRNA^Trp(CCA); (K) tRNA^Lys(CUU); (L) tRNA^Lys(UUU); (M) tRNA^Pro(CGG).

Figure 5

Summary of the tRNA introns identified in S. tokodaii strain7 [15]. Each label includes the tRNA species, the respective intron position relative to the 5' end of respective tRNA, and the lengths of the introns. Characters within parentheses indicate the nucleotide one base 5' from the intron border.

Figure 6

Overall strategy used to clone cDNAs that represent actual S. tokodaii tRNAs [15].

Figure 7

Exon-intron border regions of unspliced forms of tRNA^Glu(UUC) (A); tRNA^Glu(CUC) (B); and tRNA^Leu(GAG) (C) of S. tokodaii strain7 [15]. These are examples of introns located at positions other than position “37/38”. In (A,B), only the D-arm region is shown; In (C), only the D-arm and anticodon arm regions are shown. The arrowheads indicate the exon-intron borders determined in our previous study [15]. The anticodon sequence of tRNA^Leu(GAG) is boxed.

Figure 8

The BHB-like structure in the unspliced form of tRNA^Met(CAU-1) of S. tokodaii strain7 [15]. Only the anticodon arm and extra loop regions are shown. (A) The structure predicted by Marck and Grosjean [19]; (B) the structure inferred by in vitro cleavage of the intron with recombinant S. tokodaii EndA [15]. The exon-intron borders determined by the biochemical study [15] are indicated by arrowheads. Dashed lines in (A,B) show the cleavage sites predicted by Marck and Grosjean [19]. The anticodon sequences are boxed.