TRANSCRIPTION OF BACTERIOPHAGE T3 DNA CATALYSED BY ESCHERICHIA COLI AND T3 RNA POLYMERASES

Abstract

The transcriptional program of the virulent double-stranded DNA coliphage T3 in infected cells can be divided into two stages. Early in infection, the host (E. coli) RNA polymerase transcribes the leftmost 20% ('early', or Class I genes) of the genome to generate a polycistronic early mRNA. One of the early gene products is itself a T3 specific RNA polymerase which then copies the remaining 80% ('late', or Class II and Class III genes) of T3 DNA.;Early transcription begins at a set of 2 or 3 major promoters for E. coli RNA polymerase located at the left end of T3 DNA, terminating at a site near 20% of the genome. It was observed that in vitro, the termination efficiency increased with the KCl concentration in the reaction. Additionally, termination was affected by perturbations in the secondary structure of the nascent RNA introduced by substituting the normal bases with analogs having altered base-pairing properties. Three types of base-substitution were carried out on RNA: Guanine (G) (--->) Hypoxanthine (H), Cytosine (C) (--->) 5-Bromocytosine (5BrC), and Uracil (U) (--->) 5-Bromouracil (5BrU). Termination at 20% was abolished by G (--->) H substitution, whereas increased termination efficiency resulted from the C (--->) 5BrC change; 5BrU substitution had no apparent effect. Since H forms a less stable base-pair with C than does G, whereas 5BrC:G pairs are more stable than C:G, these observations elucidated the importance of RNA secondary structures involving G:C pairs in determining termination efficiency.;Early transcription in vitro was found to be sensitive to the E. coli termination protein, rho. In presence of rho, RNA polymerase terminates at two major sites located around 8% and 15% of the genome. Termination at these sites is salt-sensitive, rho being inactive at high (0.2M) KCl concentrations. Rho activity was remarkably influenced by base-substitutions in the nascent RNA. Thus, incorporation of H in place of G greatly stimulated (i) rho-mediated inhibition of RNA synthesis, and (ii) rho-dependent ATPase activity. Size analysis of the products revealed heterogeneous termination at or before 8% of the genome. A C (--->) 5BrC change had the reverse effect: there was no inhibition of RNA synthesis in presence of rho, or any detectable rho ATPase. 5BrU, in place of U, had marginal inhibitory effects on rho activity. The activities of rho in termination and ATP hydrolysis are therefore inversely related to the stability of secondary structure of the nascent transcripts, i.e., rho interacts effectively only with single-stranded regions of RNA.;Transcription of late T3 genes is catalyzed by a T3-specific RNA polymerase. To locate the promoters for this polymerase on the T3 genome, a detailed restriction map was first constructed. The restriction maps were used to locate the 5' termini of T3 RNA polymerase transcripts by a Southern hybridization technique. This experiment showed that, in vitro, T3 RNA polymerase initiates transcription from different regions of the genome, including the early region.;Two of the promoters (map locations: 1 m.u. and 63.9 - 69.2 m.u.) were chosen for further study. Restriction fragment transcription, RNA and DNA sequence analysis were employed to determine the nucleotide sequence around the initiation sites. The 20-bp sequence A N T T A N C G T C A C T A A A G('+1)G G A is highly conserved between the two T3 promoters. They are also homologous, especially between positions -9 and +4, to the prototype promoter sequence for T7 RNA polymerase. Since T3 and T7 RNA polymerases are specific for their respective templates, these observations raise interesting questions on the basis of promoter recognition by these two enzymes.