In vitro expression of the intron-containing gene for T4 phage thymidylate synthase

The mechanism of expression of the structural gene (td) of T4 phage thymidylate synthase, which contains a 1,017-base pair intron, was studied by employing a coupled transcription-translation system with a td containing recombinant plasmid (pKTd2) as template. The [3H]leucine-labeled protein products synthesized in this system were treated with antibody to the synthase and the resulting immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two labeled polypeptides were obtained, one with an Mr of 32,000 and the other with an Mr of 25,000. The former corresponds in molecular weight to a subunit of T4-thymidylate synthase and the other to the 183-amino acid peptide encoded by exon I, the 5'-end of the interrupted td gene. When pKTd2 restricted in exon I was used as a template, labeled immunopeptides were not detected but, when restricted in the intron region or in exon II, only the 25,000 Mr exon I product was obtained. Both peptides (Mr = 25,000 and 32,000) were synthesized when the gene was restricted downstream to exon II. Active enzyme, as measured by the tritium release assay, was shown to form about 6 min after the td gene was added to the in vitro protein synthesizing system, and followed the appearance of mature mRNA, as evidenced by S1 nuclease protection studies. The enzyme increased linearly for another 14 min in conjunction with the appearance of the Mr = 32,000 immunopeptide. The exon I product, however, preceded the Mr = 32,000 peptide, indicating that a post-transcriptional processing event may be required for mature mRNA to be formed. Measurement of the RNA products from the td gene in a transcriptional system, with labeled probes from specific regions of the td gene, provided evidence in support of an RNA processing mechanism involving intron excision and exon splicing.