Bacteriophage Lambda (Nut R): a Class of Mutants Defective for Utilization of the Lambda Gene N Antitermination Function.

Abstract

Early transcription of bacteriophage lambda initiates at two promoters, P(,L) and P(,R), and proceeds in opposite directions until stopping at two rho-dependent terminators, t(,L1) and t(,R1). The N gene lies in the left transcript, and its product apparently interacts with RNA polymerase at two sites on the DNA, nutL and nutR, enabling the polymerase molecules to then proceed through terminators located downstream from the nut sites. We devised a selection and screening procedure that enabled us to isolate and characterize (lamda)nutR phage. The selection was based on our constructing conditions whereby a lysogen harboring a temperature-inducible prophage would survive prophage induction only if transcription past terminators did not occur. Survivors were then screened to find those that had functional but unexpressed O and P genes, as measured by recombination tests, and functional and expressed N and cro genes, as measured by complementation tests. We mapped the mutation in those lysogens passing the screening procedure and chose for further study 4 lysogens whose prophage contained a mutation in the region located between the right end of the 434 immunity region and the r32 insertion. We rescued the phage from these lysogens by making use of the fact that functionally N('-) phage, presumably including nutR, can propagate in a rho(,ts15) host. This was found to hold true for the (lamda)nutR phage. As determined by fine structure genetic mapping, the mutations lie in the region containing the site previously designated to be the nutR site by virtue of sequence homology with the nutL site. The burst time of the (lamda)nutR phage varies directly with the temperature that the phage are propagated at, apparently reflecting an increased rho activity at high temperature in wild type E. coli K12. The nutR mutation apparently does not affect the ability of Ngp modified RNA polymerase to transcribe through Ngp-sensitive terminators. However, it was found that repressor-bound lambda operators act as a barrier to transcription elongation occurring in the direction of P(,L) to P(,R).