Nucleotide sequence of the primer RNA for DNA replication of filamentous bacteriophages

Abstract

We determined the nucleotide sequence of RNA synthesized in vitro by Escherichia coli RNA polymerase at the complementary-strand replication origin on the single-stranded viral DNA of bacteriophages f1 and IKe (ori-RNA) by using chain-terminating ribonucleoside triphosphate analogs. The results indicated that the start site of f1 ori-RNA synthesis is 20 nucleotides downstream from the site previously reported (K. Geider, E. Beck, and H. Schaller, Proc. Natl. Acad. Sci. USA 75:645-649, 1978) and that the RNA sequence [(5')pppAGGGCGAUGGCCCACUACGU-OH(3')] is complementary to the f1 DNA sequence from nucleotides 5736 to 5717, with minor heterogeneity at the 3' end. IKe ori-RNA had a sequence identical to that of f1 ori-RNA, except for a single base substitution, and IKe RNA was complementary to a region of IKe DNA (from nucleotides 6441 to 6422) that was homologous to the f1 sequence. Phenotypes and ori-RNA sequences in the relevant region of the genome of f1 deletion mutants were consistent with the presently determined sequence of ori-RNA. A possibility that ori-RNA synthesis is initiated by a mechanism similar to that for general transcription is suggested as a result of the new assignment of the ori-RNA start site. The double-origin plasmid assay of minus-strand origin activity, a sensitive in vivo method for detecting cis-acting elements for the initiation of DNA replication on a single-stranded DNA template, is described.