Mechanism and requirements of in vitro RNA splicing of the primary transcript from the T4 bacteriophage thymidylate synthase gene

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Abstract
The splicing of a procaryotic precursor RNA transcribed from the T4 phage thymidylate synthase (td) gene with SP6 RNA polymerase was investigated in vitro. The intron excision-cyclization reaction increased progressively to 60.degree. C. Exon ligation, though barely detectable at the lower temperatures, was greatly enhanced at 60.degree. C. Both reactions required Mg2+. The addition of guanosine to the 5'' end of an intron-exon II intermediate via a 3'',5''-phosphodiester bond was essential for the ligation of exon I to exon II. The added guanosine and the first intron-encoded uridine are subsequently lost as a dinucleotide from the 5'' end during cyclization of the linear form of the excised intron RNA. Exon ligation is intramolecular and occurs more readily in the nascent RNA molecule (cotranscriptionally) than in the finished transcript (posttranscriptionally). These data and the identification of various structural elements (P, Q, R, S, E, E'') in the td intron that are found typically in eucaryotic class I introns firmly establish the td intron as the first example of class I intron of procaryotic origin.