Genetic transcription in bacteriophage λ: Studies of λ mRNA synthesis in vivo

Abstract

Physically separated, complementary strands of λ DNA which are linearly intact, biologically active when re-annealed and free from contamination by the opposite strand have been prepared by centrifugation of denatured DNA in cesium chloride gradients in the presence of polyguanylic acid and subsequent removal of the ribonucleotide homopolymer by hydrolysis in 0.3 n-KOH at 37 °C. DNA-RNA hybridization experiments using these strands as well as the separated AT-rich‡ (right) and GC-rich (left) halves of λ DNA indicate that major shifts in the predominant direction and location of λ mRNA synthesis occur during the course of phage development. λ-Hybridizable RNA synthesized in vivo at early times after induction of λ lysogens is transcribed mainly from right to left from nucleotide sequences on the AT-rich half of the λ DNA strand having its 5′-phosphate group at the sus-A end of the genetic map. Approximately 90% of the λ-specific RNA made at late times after induction is synthesized in the opposite direction at sites located on both λ DNA halves. Studies carried out with λ mutants that are defective in early functions, or with wild-type λ under conditions where either protein synthesis or DNA replication was inhibited, indicate that transcription of the λ genome occurs in at least three stages: (1) copying of “early” genes which are under the direct control of cI repressor; (2) early gene transcription dependent upon protein synthesis and function of the N gene; and (3) (late) transcription which is dependent upon DNA replication and Q gene function. The annealing patterns of 3H-labeled λ-specific RNA synthesized by λ lysogens suggest that transcription of prophage genes occurs in divergent directions on both strands of the template. Other hybridization experiments carried out with λ RNA isolated from lysogens which have been appropriately superinfected to hyperproduce cI mRNA confirm the existence of bidirectional transcription within the λ immunity region.