Regulation of bacteriophage T4 gene expression

Abstract

The programme of bacteriophage T4 gene expression and its control by DNA synthesis and by the expression of maturation product function have been investigated in detail by the analysis of RNA, protein synthesis and the synthesis of intracellular phage. Single amber maturation-defective mutants in gene 33 are shown not to block maturation totally. Since single amber mutants can be leaky, multiple amber mutants defective in DNA synthesis and maturation product functions are used here. It is known that mutually complementary transcripts are made during T4 development. The experiments described here show that the anti-late RNA from DNA l-strand appears at two minutes after infection at 30 °C, at the same time as delayed early RNA. Anti-late RNA reaches a steady-state concentration at four minutes. It constitutes 2% of the total T4 RNA present in the 5-min RNA and it is at least as stable as the l-strand-specific phage messenger RNA. The DNA template contributing to the transcription of anti-late RNA is estimated to be at least 81% of that DNA l-strand which is complementary to the entire length of the DNA r-strand involved in the transcription of late RNA. Prereplicative r-strand transcript constitutes only 0.1% of the total prereplicative phage RNA. The increased synthesis of quasi-late RNA from the DNA l-strand starts to occur between six and ten minutes after infection. The r-strand late transcription starts between 10 and 12 minutes after infection and all the late RNA is transcribed at least once in this two-minute interval. The observation that at least 14 new protein bands (late proteins) first appear between 11 and 14 minutes is consistent with these observations on the synchronous initiation of late RNA species. Studies by other investigators using single amber DNA negative and maturation defective mutant infections show that (1) quasi-late RNA content of 20-min DO † † Abbreviations used: D0, DNA negative; MD, maturation deficient. and MD-infected cells is similar to that of 20-min am + RNA and that (2) all the DNA l-strand late RNA species are present in lower amounts in the 20-min MD RNA. Using multiple mutants in the present studies, the l-strand quasi-late RNA content in the 20-min D0 and MD RNA is found to be intermediate between that in 5 and 20-min am + RNA and the absolute requirement of maturation product function is demonstrated for the l-strand late RNA transcription. DNA synthesis is shown to be required for the initiation of l-strand late transcription and the continued synthesis of the l-strand RNA is dependent on continued replication. Although D0 and MD infections have the same phenotype as far as the synthesis of late RNA and of late proteins is concerned, the accentuation of some of the early RNA species and of at least two early proteins are shown to differ. In experiments performed with a temperature-sensitive mutant in DNA polymerase (gene 43), late transcription has been shown to begin within one minute after the temperature shift down from the non-permissive to the permissive temperature; and within two minutes of the temperature shift about two-thirds of the maximum amount of late RNA is synthesized. These results are interpreted to indicate that the initiation of late RNA synthesis begins synchronously without a lag, suggesting that the synthesis of late RNA may occur on the parental DNA strand under these conditions.