Initiation sites for discontinuous DNA synthesis of bacteriophage T7.

We have previously shown that the discontinuous replication of bacteriophage T7 DNA is primed by tetraribonucleotides (major component) or pentaribonucleotides. Both tetramers and pentamers start with pppA-C and are rich in A and C at the third and fourth nucleotides. In this study, the sites of transition from primer RNA to DNA in vivo have been located on a 340-nucleotide segment of the H strand of the T7 genome by 32P-labeling in vitro of the 5'-hydroxyl ends of DNA resulting from alkaline hydrolysis of RNA-linked T7 DNA fragments. Five strong transition sites were detected with a common sequence 5'-G-A-C-N1-N2-N3-N4-3', in which N1 was either C or A, N2 ws A, C, or G, and either N3 or N4 was the nucleotide for the switchover to DNA synthesis. We conclude that the complementary sequence 3'-C-T-G-G/T-N'2-(N'3)-5' in the template strand is the most frequently used signal for synthesis of primer RNA. Whereas primer-RNA synthesis starts at a precisely defined nucleotide, the transition to DNA synthesis varies within two nucleotides. Because the observed signal sequence would be present on a statistical basis once per 128 nucleotides, only about 10% of the existing signals are used for primer synthesis in each round of replication so that nascent fragments 1000-2000 long result. This provides an unexpected flexibility for RNA priming of DNA synthesis.