Two domains for splicing in the intron of the phage T4 thymidylate synthase (td) gene established by nondirected mutagenesis

Of 97 nondirected T4 thymidylate synthase-defective ( td ) mutations, 27 were mapped to the intron of the split td gene. Clustering of these intron mutations defined two domains that are functional in splicing, each within approximately 220 residues of the respective splice sites. Two selected mutations, td N57 and td N47, fell within phylogenetically conserved pairings, with td N57 disrupting the exon I-internal guide pairing (P1) in the 5′ domain and td N47 destabilizing the P9 helix in the 3′ domain. A splicing assay with synthetic oligonucleotides complementary to RNA junction sequences revealed processing defects for T4 td N57 and T4 td N47, both of which are impaired in cleavage at the 5′ and 3′ splice sites. Thus prokaryotic genetics facilitates association of specific residue changes with their consequences to splicing.