STARTING NUCLEOTIDE SEQUENCES OF RNA SYNTHESIZED ON REPLICATIVE FORM DNA OF COLIPHAGE FD

The replicative form DNA, with no single-strand break (RF-I DNA), was purified from Escherichia coli cells infected with a filamentous coliphage fd, and the starting nucleotide sequences of RNA formed by E. coli RNA-polymerase under the direction of RF-I DNA as template were analysed. Incorporation of four nucleoside [γ-32P]triphosphates into the 5′-end of RNA was studied, and it was found that RNA chains synthesized on RF-I DNA were initiated predominantly with ATP- and GTP-ends. The ratio of the ATP-end to GTP-end was roughly 1:1.6 to 2.0 under the conditions used. RNA was synthesized in the reaction mixture containing [γ-32P]ATP and [3H]ATP. The labelled RNA was hydrolysed with pancreatic and T1 RNases, and radioactive fragments produced were analysed. The main terminal fragment identified was pppApUpGp. RNA synthesized with [γ-32P]GTP and [3H]GTP was hydrolyzed with pancreatic RNase, and resulting radioactive fragments were analysed. The predominant terminal fragment produced was pppGpUp. In order to identify the third nucleotide of RNA starting with the GTP-end, RNA was synthesized with nucleoside [α-32P]triphosphates, and nearest-neighbour analysis was carried out. A and U were found in the third position. It was concluded that at least three species of RNA starting with the following sequences were synthesized under the direction of RF-I DNA of phage fd: pppApUpGp-----, pppGpUpAp-----, pppGpUpUp-----. © 1969.