Controls and polarity of transcription during bacteriophage T4 development

Hybridization and hybridization-competition experiments, using the separated l and r strands of bacteriophage T4 DNA and 3H-labeled RNA's coded by T4 and its mutants, permit one to study the transcriptional controls for various phagespecific RNA species. In the present study, the T4-coded RNA's have been classified according to: (i) orientation of transcription; (ii) time of appearance; (iii) requirement for protein and DNA synthesis and for expression of the gene 33 and 55 functions; and (iv) true-early or quasi-late behavior, as defined by the respective decrease or increase in the early appearing RNA species during the second half of the viral eclipse period. The “immediate-early” messenger RNA's, produced after phage infection in the absence of protein synthesis, are transcribed counterclockwise from the l strand of T4 DNA. The “delayed-early” RNA is transcribed predominantly from the l strand, whereas the “true-late” RNA, the synthesis of which starts later than five minutes after infection, is mainly transcribed clockwise from the r strand, i.e. with the same orientation as T4 DNA replication. There is only a small difference in the 20-minute l-strand transcription between the wild-type T4 and its mutants blocked in DNA synthesis (D0; genes 42 and 43) or in the late functions (MD, maturation defective) controlled by genes 33 and 55, as revealed by hybridization-competition experiments. Also, 5-minute unlabeled T4-coded RNA competes with almost 90% of the labeled 20-minute RNA complementary to the l strand, indicating that, at most, 10 to 12% of the 20-minute l-specific RNA belongs to the true-late class. Experiments employing r strands show that the 20-minute RNA of the D0 and MD mutants is quite deficient in RNA species coded by wild-type T4 at 20 minutes after infection. Similarly, under non-permissive conditions, mutations in gene rII and in genes 46 and 47, the latter two controlling nuclease activity, depress the r-strand transcription, but mutation in gene 30, which controls T4 ligase, does not have such an effect. The influence of chloramphenicol inhibition at various stages of T4 development on the l and r strand transcription was also evaluated. The activity of the gene 55 product and of DNA replication are continuously required for efficient r strand transcription.