A novel group I intron-encoded endonuclease specific for the anticodon region of tRNAfMet genes

Abstract

Open reading frames (ORFs) are frequently inserted into group I self-splicing introns. These ORFs encode either maturases that are required for splicing of the intron or DNA endonucleases that promote intron mobility. A self-splicing intron in the tRNAfMet gene of Synechocystis PCC 6803, which has been proposed to have moved laterally within the cyanobacteria, contains an ORF that is unrelated to known intronencoded endonucleases or maturases. Here, using an in vitro transcription-translation system, we show that this intronic ORF encodes a double-strand DNA endonuclease, I-Ssp68031. I-Ssp68031 cleaves each strand of the intronless tRNAfMet gene adjacent to the anticodon triplet leaving 3 bp 3′ extensions and has no activity at intron-exon boundaries. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a partially palindromic 20 bp region encompassing the entire anticodon stem and loop of the tRNAfMet gene. I-Ssp68031 represents a novel intron-encoded DNA endonuclease and is the first example of a chromosomally encoded group I intron endonuclease in bacteria.