Structure, function, and evolution of the tRNA endonucleases of Archaea: an example of subfunctionalization

Abstract

We have detected two paralogs of the tRNA endonuclease gene of Methanocaldococcus jannaschii in the genome of the crenarchaeote Sulfolobus solfataricus. This finding has led to the discovery of a previously unrecognized oligomeric form of the enzyme. The two genes code for two different subunits, both of which are required for cleavage of the pre-tRNA substrate. Thus, there are now three forms of tRNA endonuclease in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaea and the Nanoarchaea. The last-named enzyme, arising most likely by gene duplication and subsequent "subfunctionalization," requires the products of both genes to be active.

Fig. 1.

Primary sequences and secondary structures of tRNA endonuclease subunits. A sequence alignment of tRNA endonuclease subunits is presented as two separate groups as described in the text. Sequences belonging to the structural subunit class are colored cyan, and sequences belonging to the catalytic subunit are colored green. Conserved residues are in red, and similar residues are in blue. Cyan columns indicate positions that are conserved among the catalytic subunits. Green columns highlight positions that are conserved among the structural subunits. Yellow columns indicate residues that are conserved in both groups. Secondary structure elements, as determined from the crystallographic structure of METJA, are indicated on top of the sequence of NR_ARCFU. Red arrows indicate β-strands, and green cylinders represent α-helices. The plus signs at the bottom of the alignment indicate the conserved catalytic triad Tyr-115/His-125/Lys-156.

Fig. 2.

Structural analysis. (A) Results of the multiple sequence alignment mapped onto the three-dimensional structure of a METJA monomer (1A79). Residues conserved in the structural subunits are colored cyan; residues conserved in the catalytic subunit are in green. (B) Sequence conservation mapped onto the structure of the tetramer of METJA (1A79).

Fig. 3.

tRNA endonuclease activity assays. (A) Pre-tRNA^archaeuka consists of two regions derived from yeast pre-tRNA^Phe (nucleotides 1–31 and 38–76) joined by a 25-nt insert that corresponds to the bulge–helix–bulge motif of archaeal tRNA^Trp. (B) Pre-tRNA archaeuka was incubated with four different recombinant proteins. The cleavage products were analyzed by electrophoresis on 10% polyacrylamide (29:1)/8 M urea gel, followed by autoradiography. The identification of the reaction products is indicated. Lane 1, control (no enzyme added); lane 2, endonuclease heterotetramer of SULSO; lane 3, endonuclease homodimer of ARCFU; lane 4, His-6-tagged NR_ARCFU domain alone; lane 5, NR_ARCFU coexpressed with CR_ARCFU domain. (C) Pre-tRNA archaeuka was incubated with five different recombinant purified proteins. Cleavage products were analyzed as in B. The identification of the reaction products is indicated. Lane 1, α_SULSO subunit; lane 2, α_SULSO coexpressed with the β_SULSO subunit; lane 3, β_SULSO subunit; lane 4, β_SULSO subunit coexpressed with the α_SULSO subunit; lane 5, control (no enzyme added); lane 6, METJA endonuclease. * indicates His-6 tagging.

Fig. 4.

Schematic representation of the phylogenetic tree of Archaea (adapted from ref. 21). Each rectangle surrounds species sharing the same endonuclease architecture. The subunits are named according to Fig. 5. The duplication events are indicated by an arrow.

Fig. 5.

Model of the tRNA splicing endonucleases of METJA, ARCFU, and SULSO. (A) model of the METJA homotetramer. Several important structural features discussed in the text are indicated: loop L10 (cyan triangle), the C-terminal β10 strand (arrow), and the conserved catalytic triad residues Tyr-115, His-125, and Lys-156 (green pentagon). (B) Proposed subunit arrangement of the ARCFU endonuclease. The NR lacks two of the three putative active-site residues. It does, however, contain many of the features of the C-terminal domain, which are important for structural arrangements of the enzyme: in particular, the L10 sequence. The C-terminal repeat contains all of the sequence features of the METJA enzyme. Black lines represent the polypeptide chain connecting the CRs and NRs. (C) A proposed model for the SULSO endonuclease. The α-subunit of the SULSO enzyme contains the conserved catalytic triad (green pentagon) and the C-terminal β10 strand (arrow). The β-subunit of the SULSO enzyme contains loop L10 (cyan triangle) and the C-terminal β10 strand (arrow).