Studies on bacteriophage fd DNA. II. Localization of RNA initiation sites on the cleavage map of the fd genome.

In an in vitro RNA synthesizing system, a single size of A-start RNA and three different sizes of G-start RNA are predominantly transcribed on the doubly closed replicative form (RFI) DNA of phage fd. When the RFI DNA was cleaved into three fragments (HinH-A, HinH-B and HinH-C) by a restriction endonuclease from Haemophilus influenzae H-I, the A-start RNA was predominantly initiated on HinH-B and the three G-start RNAs on HinH-A. RFI DNA was further cleaved into smaller pieces by two other restriction endonucleases from H. aphirophilus and H. gallinarum. Upon mixing the digests with RNA polymerase, two specific fragments derived from HinH-A were bound to the polymerase with GTP present. G-start RNA was efficiently initiated on the fragments isolated by this procedure. On the basis of these observations and estimates of the size of RNA formed on each fragment, the initiation sites for major RNA species were localized on the cleavage map of the phage fd genome previously constructed.