Figure 2

Thermotargetron method for gene targeting in thermophiles. A targetron expression plasmid with a strong promoter is used to express a thermostable, approximately 0.8-kb group II intron RNA (TeI3c) and a group II intron reverse transcriptase (TeI4c RT; denoted RT) from the thermophilic cyanobacterium Thermosynechococcus elongatus[25, 26]. The intron RNA is expressed as a precursor with short flanking exon sequences (denoted E1 and E2) and is spliced from this precursor with the assistance of the intron-encoded RT to yield a ribonucleoprotein (RNP) complex in which the RT is bound to the excised intron lariat RNA. Group II intron RNPs recognize DNA target sites primarily by base pairing of sequence motifs in the intron RNA to the DNA target sequence, with only a small contribution from the intron-encoded RT. The intron RNA then uses its catalytic (ribozyme) activity to insert into the top strand of the DNA target site between target exon sequences (E1’ and E2’) (A), while the DNA endonuclease activity of the RT is used to cleave the bottom strand and the nicked DNA is used as a primer for reverse transcription of the inserted intron RNA (B). The resulting intron cDNA is integrated into the genome by host enzymes (C). Because the DNA target site is recognized mainly by base pairing of the intron RNA, the targetron can be programmed to insert into desired sites simply by modifying the base-pairing motifs in the intron RNA, taking into account the small number of target site nucleotide residues recognized by the RT. The inserted targetron contains multiple stop codons in all three reading frames, resulting in the expression of truncated proteins. Targetron methods are described in detail in [23].