To explore the basis for the template specificities of the bacteriophage T3 and T7 RNA polymerases (EC 2.7.7.6), we determined the nucleotide sequences of six promoters recognized by the T3 RNA polymerase and compared them with the previously determined promoter sequences recognized by the bacteriophage T7 RNA polymerase. Recombinant plasmids containing random Hpa H and Taq I fragments of T3 DNA were screened for T3 promoter activity in vitro in a transcription assay using purified T3 RNA polymerase. Five promoters for the T3 RNA polymerase were identified in this manner and their sequences were determined; the sequence of an additional promoter was determined directly from a genomic DNA fragment. In five of the T3 promoters an identical 16-base-pair sequence (A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A) extends from -12 to +4 (initiation occurring with GTP at + 1); this sequence is preceded by a 6-base-pair A+T region. The remaining promoter contains an inserted C at position -1 and an A at the +1 position. The sequence of the 5' end of the RNA transcript from the latter promoter confirms that transcription is initiated with ATP at the +1 position. Previously, late T3 or T7 transcripts had not been found to initiate with ATP. The highly conserved T3 promoter sequence was compared to the T7 promoter consensus sequence. The fundamental difference between the two kInds of phage promoters is the occurrence of G-A at positions -11 and -10 in the T7 promoter, whereas there is a single C at position -10 in the T3 promoter.Bacteriophages T3 and T7 have been well characterized, and they have provided attractive model systems for studies of transcription control (1-3). During infection the genomes of these viruses are transcribed successively by two different RNA polymerases (EC 2.7.7.6). Initially, the early genes are transcribed by the host (Escherichia coli) RNA one promoter recognized by the T3 RNA polymerase was recently reported (8). In this paper, we describe the identification and characterization of six promoters recognized by the T3 RNA polymerase. By comparing the consensus sequences of the T3 and T7 promoters, we have identified a single region that appears to be critical for the specificity of RNA polymerase recognition. MATERIALS AND METHODSIsolation and Cleavage of DNA. Bacteriophage T3 (Hausmann strain) DNA and plasmid pBR322 DNA were isolated as described (9, 10). Conditions of cleavage with restriction endonucleases were those recommended by Davis et aL (11).Cloning and Identification of T3 DNA Fragments Having Promoter Activity. pBR322 DNA was digested with Cla I, treated with calf intestine alkaline phosphatase, extracted with phenol, mixed with Taq I or Hpa II fragments of T3 DNA at a molar ratio of 4:1 (plasmid to fragments), and incubated overnight in the presence of T4 DNA ligase at 14WC (11). Competent HMS174 cells (10, 12) were transformed with the recombinant DNA, and ampicillin-resistant tetracycline-sensitive colonies were isolated and propagated overnight in 2 ml of M9-T broth (13) containing...

The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.

The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, Bgm, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7.T3 and T7 are closely related bacteriophages which share many features but differ in interesting and potentially significant ways (7). For example, both viruses elaborate phage-specified RNA polymerases during infection which, although they are similar, do not transcribe the heterologous DNA efficiently (3, 5). Although the two viruses have similar genetic organizations (1, 7), a detailed description of the physical structure of the genome (e.g., by restriction analysis) existed only for bacteriophage T7 (10, 12). Restriction maps of both viruses would be useful for locating and comparing regulatory signals in the DNAs and could serve as a basis for estimating the evolutionary divergence of the two viruses. In this paper, we describe the derivation of a restriction map for bacteriophage T3 DNA and compare it with the map of T7 DNA. MATERIALS AND METHODSBacterial and phage strains and preparation of DNA. Bacteriophage T3 Hausmann (15) and Escherichia coli B were from the laboratory of E. K. F. Bautz. Deletion mutants of T3 (16) were obtained from F. W. Studier.DNA was isolated from CsCl-purified virus particles as previously described (9). For [nP]DNA, lysates were prepared as previously described (9), and the phage particles were harvested by centrifugation at 35,000 x g for 80 min, suspended in buffer (10 mM Tris, pH 7.5,5 mM Mg9l2, 50 mM NaCI), and clarified by centrifugation at 12,000 x g before isolation of DNA.Cleavage of DNA and electrophoresis of fragments. AvaI, XbaI, KpnI, BglII, HindIII, MboI, and HpaI (11) were purchased from Bethesda Research Laboratories, Rockville, Md. MboI has the same specificity as DpnII, which was used previously to map T7 DNA (10, 11). The conditions of cleavage were those recommended by the supplier. For double digestions, the first enzyme was inactivated by the addition of 10 mM EDTA, followed by heating to 650C for 5 min.The DNA was precipitated with ethanol and sodium acetate, washed with 70% ethanol, dried in vacuo, and taken up in buffer suitable for cleavage by the second enzyme. DNA fragments were analyzed by electrophoresis through agarose or polyacrylamide gels, as previously described (10,12).Isolation ofindividual fragments from agarose gels. Individual fragments were resolved by electrophoresis through 0.6% agarose gels and visualized by sta...

Promoter locations for the T3 RNA polymerase on the physical map of T3 DNa have been determined. Through the use of conditions favoring the synthesis of RNA from the class II region, an agarose-formaldehyde gel system which improves the resolution of high molecular weight RNAs, and template DNA that was cut by one of several restriction endonucleases prior to transcription, seventeen promoter locations for the T3 RNA polymerase have been mapped. Ten promoters have been identified in the class II region and one promotor has been identified in the class II region and one promotor has been identified in the early (class I) region. The locations of previously mapped class III promoters and the internal termination signal for the T3 RNA polymerase have been mapped more precisely than in previous reports. The resulting transcription map demonstrates a striking similarity to the transcription map of bacteriophage T7.

The early genes of bacteriophages T7 and T3 are transcribed by E. coli RNA polymerase from a cluster of three promoters that lie near the left end of the genome (1 ,2). We have sequenced the early control region of T3 DNA and have compared it with the corresponding region of T7 DNA (Fig. 1). Three potential promoters for the E. coli RNA polymerase (designated Al, A2 and A3) are apparent from their homology to the Al, A2 and A3 promoters of T7, as well as by their homology to the consensus sequence for other E. coli promoters (4).