The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the X PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.During bacteriophage T7 infection, the right-most 80% of the genome is transcribed by a phage-encoded RNA polymerase, the product of gene I (Fig. 1) (1). In contrast to the multisubunit RNA polymerases of bacteria and eukaryotes, T7 RNA polymerase is a single polypeptide of molecular weight 98,800 (2, 3). The enzyme is specific for its own promoters, a conserved 23-base-pair (bp) sequence (4-6).T7 RNA polymerase is present in relatively low amounts in T7-infected cells, constituting 0.1% of the cellular protein.To facilitate studies on its role in the initiation of T7 DNA replication (7), we have placed its gene on a plasmid under the control of the A PL promoter. When induced, T7 RNA polymerase constitutes 20% of the soluble protein, permitting a simple purification of the enzyme to homogeneity. Davanloo et al. (8) have also described the purification of T7 RNA polymerase from cells overexpressing the cloned T7 gene 1.A logical extension of these studies is to exploit the specificity of T7 RNA polymerase for its promoters to express other cloned genes. Transcription by Escherichia coli RNA polymerase can be inhibited selectively by the addition of rifampicin. Here, we use the T7 RNA polymerase/promoter system to overproduce bacteriophage T7 gene 5 protein, a subunit of the T7 DNA polymerase (Fig.

DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle, while achieving error frequencies in the range of 10(-5) to 10(-6). Here we present a 2.2 A crystal structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer-template and a nucleoside triphosphate in the polymerase active site. The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases.

A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range ofa few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.The dideoxynucleotide method for DNA sequencing is based on the ability of a DNA polymerase to extend a primer, annealed to the template that is to be sequenced, until a chain-terminating nucleotide is incorporated (1). The resulting series of unique fragments are separated by polyacrylamide gel electrophoresis. Ideally, a DNA polymerase used for sequencing should (i) have high processivity and a rapid rate of nucleotide incorporation, (ii) lack exonuclease activity, and (iii) not discriminate against nucleotide analogs.(i) Processivity is the ability of a single enzyme molecule to polymerize nucleotides on a DNA chain without dissociating (2). Low processivity can lead to a background of artifactual bands that result from terminations by random dissociation rather than by the incorporation of a chain-terminating nucleotide. A slow rate of incorporation can accentuate pause sites. (it) An exonuclease activity can cause variability in the intensity of radioactive fragments by increasing the probability of the incorporation of a chain-terminating nucleotide at positions where there is increased activity. It can enhance pausing at sequences with secondary structure and increase the discrimination against analogs. (iii) A number of nucleotide analogs are useful in improving the resolution of DNA sequencing gels: 2',3'-dideoxynucleoside 5'-triphosphates (ddNTPs) are used to specifically terminate chains (1), 2'-deoxynucleoside 5'-[a-thio]triphosphates are used to label the fragments with 35S (3), and analogs of dGTP (dc7GTP and dITP) are used to remove gel compressions that result from base-pairing (4-6).The DNA polymerases currently used for sequencing are the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) (1) and avian myeloblastosis virus (AMV) reverse transcriptase (7). The large fragment of DNA polymerase

Helicases that unwind DNA at the replication fork are ring-shaped oligomeric enzymes that move along one strand of a DNA duplex and catalyze the displacement of the complementary strand in a reaction that is coupled to nucleotide hydrolysis. The helicase domain of the replicative helicase-primase protein from bacteriophage T7 crystallized as a helical filament that resembles the Escherichia coli RecA protein, an ATP-dependent DNA strand exchange factor. When viewed in projection along the helical axis of the crystals, six protomers of the T7 helicase domain resemble the hexameric rings seen in electron microscopic images of the intact T7 helicase-primase. Nucleotides bind at the interface between pairs of adjacent subunits where an arginine is near the gamma-phosphate of the nucleotide in trans. The bound nucleotide stabilizes the folded conformation of a DNA-binding motif located near the center of the ring. These and other observations suggest how conformational changes are coupled to DNA unwinding activity.