Initiation of coliphage lambda replication, lit, oop RNA synthesis, and effect of gene dosage on transcription from promoters PL, PR, and PR

Abstract

The requirements for initiation of lambda (λ) bidirectional replication were examined. About five initiation events occur from induced nonexcising λ prophage, compared with nearly fourfold this level for excision-competent prophage. Replication from excised prophage is higher from the int to N gene interval than from tof-P, suggesting some of the late or circle mode replication is initiated unidirectionally to the left of the origin ( ori) site. The initiation of bidirectional (early) replication and λ prophage excision are necessary predecessors for initiation of the late mode of circle replication. The Escherichia coli dnaB, dnaE, dnaG gene products are required for initiation of bidirectional λ replication and the products of genes dnaC and PolA are necessary for normal λ DNA synthesis. The combination of dnaC and hsmts prevents initiation of λ replication at the nonpermissive temperature. The products of λ genes O and P and an active ori site are also required for early replication. Two ori − mutations and a nonsense mutation in O have severe and moderate polar effects on righward transcription. One of the ori − mutations is a deletion of 15 nucleotides within gene O (Denniston-Thompson et al., 1977). The leftward P L- att transcription is independent of gene dosage effects, whereas the rightward transcription from P R and P R is slightly and very dependent on template concentration. Amplification of the synthesis of the short leftward lit and oop transcripts requires the same E. coli and λ gene products necessary for initiation of λ bidirectional replication, except for the product of dnaE. The coordinate stimulation of lit, oop transcription occurs in both the presence and absence of measurable initiation of λ DNA synthesis, or when the extent of λ DNA synthesis is reduced. This observation suggests the transcription of both oop and lit is positively regulated, rather than the alternative model in which the amplification of their transcription is a consequence of increasing DNA templates. It also suggests the on site is “activated” prior to the initiation of λ DNA synthesis. Models for action of the host and phage initiation factors in both replication and lit, oop transcription include either the recognition of common features of ori and P O, or participation in an activation event at ori which could change its secondary structure and in turn influence the stability and level of oop transcription from P O. Two models were proposed to explain the requirement of the initiation factors and active ori site for stimulation of lit synthesis which occurs 2000 nucleotides downstream from oop with no detectable transcription from the intervening DNA segment.