Characterization of the intron in the phage T4 thymidylate synthase gene and evidence for its self-excision from the primary transcript

The td gene contains a 735 bp open reading frame within its 1017 bp intron. A 12 nucleotide stretch may form a stable secondary structure with the putative Shine-Dalgarno sequence of the intron open reading frame and thus impair its translation. SP6 RNA polymerase transcripts of the td gene synthesized in vitro at 40°C encompass a 2.7 kb primary transcript, a 1.7 kb mRNA, and a 1 kb intron RNA. The excised intron RNA consisted of linear and cyclized forms. RNAase H studies and resistance of the cyclized intron to linearization by HeLa cell debranching enzyme suggest it to be circular. Self-splicing of isolated td primary transcript occurred only marginally at 28°C, but increased progressively to 50°C, and required the presence of both Mg⁺⁺ and a guanosine cofactor. An internal guide sequence is evident which may align the 5′ splice site with the 3′ end, presumably for precise exon ligation.