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Gene regulation at the right operator (OR) of bacteriophage λ

1980; Elsevier BV; Volume: 139; Issue: 2 Linguagem: Inglês

10.1016/0022-2836(80)90303-4

1089-8638

Barbara J Meyer, Russell Maurer, Mark Ptashne,

Protein purification and stability

The right operator of bacteriophage λ, OR, contains three sites, each of which is recognized by two regulatory proteins. These proteins are the λ repressor, the product of the cI gene, and the cro gene product. The interactions of these proteins with OR results in a variety of regulatory effects on transcription from the adjacent promoters PR and PRM. One purpose of this and the accompanying papers is to determine the roles played by each operator site in mediating these effects. The experiments described herein analyse the regulation of PR and PRM in vivo as a function of λ repressor or cro protein concentration. To accomplish this we utilized two types of hybrid operons constructed by recombination in vitro. One type, carried on a phage vector, bears the lacZ gene fused either to the λ PR or the λ PRM. In cells lysogenic for one of these phages, the level of β-galactosidase is a measure of the activity of PR or PRM. In the other type of hybrid operon, carried on a plasmid, a λcI or cro gene is fused to the lac promoter and synthesis of the λ regulatory protein is controlled by the lac repressor. Cells bearing one of these plasmids synthesize increased amounts of λ repressor or cro in response to increased amounts of IPTG. In a typical experiment, therefore, we measured the synthesis of β-galactosidase, as a function of IPTG concentration, in a cell bearing one of the phage vectors (as prophage) plus one of the plasmids. The roles of the OR sites in regulating PR and PRM were revealed by studying the behavior of mutants, some newly isolated, bearing sequence changes in OR. All but one of these mutations is located in OR1, OR2, or OR3, and each decreases the affinity of the corresponding site for the regulatory proteins. The other, prmup-1, is located between OR2 and OR3. Whereas wild-type PRM functions efficiently only in the presence of repressor, prmup-1 allows initiation to occur at PRM at high frequency in the absence of repressor. Our main results may be summarized as follows. (1) Occupation of OR1 or OR2 (or both) by either protein represses PR. (2) Occupation of OR3 by either protein represses PRM. (3) Repressor bound only to OR2 stimulates PRM. In a lysogen, PR is repressed and PRM is stimulated because repressor is bound predominantly to OR1 and OR2 but rarely to OR3. In contrast, as the concentration of cro protein accumulates in vivo, first PRM is turned off as OR3 is occupied and then PR is turned off as OR1 and OR2 are occupied. We show that these facts explain the contrasting physiological effect of these two regulatory proteins. We present a molecular model to explain the mechanism of each of these regulatory effects which takes into account the rules describing co-operative binding of repressor to sites in OR (Johnson et al., 1979).