TRANSCRIPTION OF BACTERIOPHAGE-PHIX174 INVITRO - SELECTIVE INITIATION WITH OLIGONUCLEOTIDES

Transcription in vitro of .vphi.X174 RFI [double-stranded closed circular supercoiled replicative form] DNA with Escherichia coli RNA polymerase yields 3 major RNA species of MW 1.87, 1.60 and 1.34 .times. 106, the largest of these corresponding to 1 round of transcription around the DNA circle. There are also 7 minor species with MW ranging from 0.07-0.84 .times. 106 which account for .apprx. 10-20% of the total incorporation of nucleotides into RNA. When rho factor is present in the reaction mixture the 3 largest RNAs are no longer made, and instead RNAs of MWs 1.00, 0.70, 0.42, 0.35 and 0.07 .times. 106 are synthesized. By initiating synthesis at specific promotor sites with oligonucleotide primers and examining the RNA products on polyacrylamide gels, all of these RNA species were mapped, and the existence of shared sites of initiation and termination were established. Three of the RNAs are artifacts of the synthesis conditions used. The others result from the combined action of 3 promotors, 2 rho-independent termination sites and 2 rho-dependent termination sites. RNA molecules initiated from 2 of the promotors begin with 5''-terminal ATP; from the other promotor they begin with 5''-terminal GTP.