Abstract
We have characterized a homodimeric tRNA endonuclease from the euryarchaeota Ferroplasma acidarmanus (FERAC), a facultative anaerobe which can grow at temperatures ranging from 35 to 42 °C. This enzyme, contrary to the eukaryal tRNA endonucleases and the homotetrameric Methanocaldococcus jannaschii (METJA) homologs, is able to cleave minimal BHB (bulge–helix–bulge) substrates at 30 °C. The expression of this enzyme in Schizosaccharomyces pombe (SCHPO) enables the use of its properties as effectors by inserting BHB motif introns into hairpin loops normally seen in mRNA transcripts. In addition, the FERAC endonuclease can create proteins with new functionalities through the recombination of protein domains.
Highlights
Nature employs various mechanisms to remove introns from mRNA, tRNA, and rRNA
The expression of this enzyme in Schizosaccharomyces pombe (SCHPO) enables the use of its properties as effectors by inserting BHB motif introns into hairpin loops normally seen in mRNA transcripts
In Archaea, unlike bacteria and eukaryotes, all introns, whether in pre-tRNA or elsewhere, use an intron excision mechanism based solely on the tRNA-splicing endonuclease and tRNA ligase [10,11,12,13]. This property is due to the ability of the archaeal tRNA endonuclease to recognize two distinct motifs: the first contains a 2 or 3 nt bulge separated by a 4 bp helix (BHB motif), and the second contains a 3 nt bulge and an internal loop separated by a 4 bp helix (BHL-like motif), regardless of the presence of a tRNA mature domain [14,15]
Summary
Nature employs various mechanisms to remove introns from mRNA, tRNA, and rRNA. In bacteriophages, bacteria, chloroplasts and mitochondria, self-splicing group I and II introns are found [1,2,3]. In Archaea, unlike bacteria and eukaryotes, all introns, whether in pre-tRNA or elsewhere, use an intron excision mechanism based solely on the tRNA-splicing endonuclease and tRNA ligase [10,11,12,13] This property is due to the ability of the archaeal tRNA endonuclease to recognize two distinct motifs: the first contains a 2 or 3 nt bulge separated by a 4 bp helix (BHB motif), and the second contains a 3 nt bulge and an internal loop separated by a 4 bp helix (BHL-like motif), regardless of the presence of a tRNA mature domain [14,15]. The FERAC endonuclease is a promising method for synthetic biology and might be used to build chimera combinations of diverse transcripts
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