Chlamydomonas reinhardtii selenocysteine tRNA[Ser]Sec

Abstract

Eukaryotic selenocysteine (Sec) protein insertion machinery was thought to be restricted to animals, but the occurrence of both Sec-containing proteins and the Sec insertion system was recently found in Chlamydomonas reinhardtii, a member of the plant kingdom. Herein, we used RT-PCR to determine the sequence of C. reinhardtii Sec tRNA[Ser]Sec, the first non-animal eukaryotic Sec tRNA[Ser]Sec sequence. Like its animal counterpart, it is 90 nucleotides in length, is aminoacylated with serine by seryl-tRNA synthetase, and decodes specifically UGA. Evolutionary analyses of known Sec tRNAs identify the C. reinhardtii form as the most diverged eukaryotic Sec tRNA[Ser]Sec and reveal a common origin for this tRNA in bacteria, archaea, and eukaryotes.

FIGURE 1.

Fractionation of C. reinhardtii tRNA. ⁷⁵Se-selenocysteyl-tRNA^[Ser]Sec was obtained by labeling C. reinhardtii cells, extracting tRNA, mixing it with a larger amount of total C. reinhardtii tRNA, chromatographing the mixture over an RPC-5 column, pooling column fractions from the two peaks of ⁷⁵Se-selenocysteyl-tRNA^[Ser]Sec as shown in the figure, and preparing the pooled fractions for sequencing as given in Materials and Methods. (Closed circles) ⁷⁵Se radioactivity; (closed diamonds) absorbance at 260 nm.

FIGURE 2.

Sequences of C. reinhardtii Sec tRNA and other RNAs. (A) Cloverleaf model of Sec tRNA^[Ser]Sec shown in a 9/4 representation (see text). The base at position 58 was determined to be T, but this position is shown as an A for reasons discussed in the text. The GenBank accession number for the nucleotide sequence of Sec tRNA^[Ser]Sec is AY268554. (B) Sequences of other tRNAs. The upper six RNAs—Arg, Asp, Cys, Pro, Thr-1, and Thr?—are new sequences; the lower three—Asn, Thr-2, and Trp—which are designated with *, were reported previously (Yu et al. 1992; Maul et al. 2002). The sequence designated Thr? is an unknown RNA, but possibly a Thr tRNA (see text), and the sequences of the three previously reported tRNAs match those shown in the figure with the exception of the bracketed base at position 16 in Thr-2 tRNA (see text). Some of the sequences, indicated by #, are partial. Each tRNA sequence is aligned at the anticodon, which is in italics and underlined. The GenBank accession numbers for the nucleotide sequences of the new RNAs are AY268556 (Arg), AY268558 (Asp), AY268559 (Cys), AY268555 (Pro), AY268557 (Thr-1), and AY268560 (Thr?). (C) Number of times each tRNA clone was sequenced among 47 independently selected bacterial colonies from each fraction (see Fig. 1 ▶).

FIGURE 3.

Multiple alignment of known Sec tRNAs and/or Sec tRNA genes. (A) Sequences from eukaryotes. Animal Sec tRNAs^[Ser]Sec were aligned with C. reinhardtii Sec tRNA^[Ser]Sec based on secondary structure (see cloverleaf model in Fig. 2A ▶). The sources of animal Sec tRNA^[Ser]Sec sequences are summarized elsewhere (for review, see Hatfield et al. 1999). (B) Sequences from C. reinhardtii, archaea, and bacteria. Prokaryotic Sec tRNAs were aligned with C. reinhardtii Sec tRNA^[Ser]Sec based on secondary structure (see above). Sources of prokaryotic sequences are given in the legend to Figure 4 ▶. The Sec anticodon of each tRNA is shown in italics. Base positions shown with a black background indicate conserved sites, those in an intermediate background, a single evolutionary change, those in a light background, two evolutionary changes, and those not shaded, more than two evolutionary changes.

FIGURE 3.

Multiple alignment of known Sec tRNAs and/or Sec tRNA genes. (A) Sequences from eukaryotes. Animal Sec tRNAs^[Ser]Sec were aligned with C. reinhardtii Sec tRNA^[Ser]Sec based on secondary structure (see cloverleaf model in Fig. 2A ▶). The sources of animal Sec tRNA^[Ser]Sec sequences are summarized elsewhere (for review, see Hatfield et al. 1999). (B) Sequences from C. reinhardtii, archaea, and bacteria. Prokaryotic Sec tRNAs were aligned with C. reinhardtii Sec tRNA^[Ser]Sec based on secondary structure (see above). Sources of prokaryotic sequences are given in the legend to Figure 4 ▶. The Sec anticodon of each tRNA is shown in italics. Base positions shown with a black background indicate conserved sites, those in an intermediate background, a single evolutionary change, those in a light background, two evolutionary changes, and those not shaded, more than two evolutionary changes.

FIGURE 4.

Phylogenetic tree of known Sec and Trp tRNAs. Sequences used to generate the tree were extracted from nonredundant and microbial databases at NCBI or were obtained as described in Fig. 3 ▶ and its legend. Nucleotide sequence alignments were generated using the PileUP program. Distances between sequences were calculated from these alignments by the DISTANCE program with the Kimura distance-measuring methods. Finally, unrooted phylogenic trees were visualized by the GrowTree program. The trees were used to trace the evolutionary origin of Sec tRNAs.