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Bacteriophage T7 RNA polymerase (T7 RNAP) is known to be one of the simplest enzymes catalyzing RNA synthesis. In contrast to most RNA polymerases known, this enzyme consists of one subunit and is able to carry out transcription in the absence of additional protein factors. Owing to its molecular properties, the enzyme is widely used for synthesis of specific transcripts, as well as being a suitable model for studying the mechanisms of transcription. In this minireview the recent data on the structure and mechanism of T7 RNAP, including enzyme-promoter interactions, principal stages of transcription, and the results of functional studies are discussed.z 1998 Federation of European Biochemical Societies.Key words: T7 RNA polymerase; Transcription; Enzyme-promoter interaction; Initiation; Elongation; Mutagenesis; Mechanism Basic properties of T7 RNA polymeraseBacteriophage T7 RNA polymerase (T7 RNAP) is known to be one of the simplest enzymes catalyzing RNA synthesis. In contrast to most RNAPs known, this enzyme (as well as those encoded by bacteriophages T3, SP6 and K11 [1,2]) is composed of one subunit. T7 RNAP transcribes late genes of bacteriophage T7 in the absence of additional protein factors. Owing to its molecular properties, the enzyme is widely used as a tool for synthesis of speci¢c transcripts, as well as being a suitable model for studying the mechanisms of transcription [3]. Recent years have seen a substantial progress in our understanding of the structure and mechanism of T7 RNAP [3,4]. This review describes current structural and functional information on this enzyme.T7 RNAP was ¢rst isolated from bacteriophage T7-infected Escherichia coli cells in 1968 [5]. The polypeptide chain of the enzyme consists of 883 amino acid (aa) residues (MW 98 092 Da) [6]. T7 RNAP is structurally related to the members of a superfamily of nucleotide polymerases that includes singlesubunit DNAPs and RNAPs such as E. coli DNAP I and reverse transcriptases. X-ray studies have demonstrated a marked resemblance between the three-dimensional structures of T7 RNAP and many DNAPs (Fig. 1) [7^9]. Thus, despite the almost complete lack of sequence homology, T7 RNAP and Klenow fragment of E. coli DNAP I demonstrate a very high structural similarity: when polymerization domains of these enzymes are superimposed, all K-helices and L-strands (except one) in the two structures correspond to each other. The shapes of these domains resemble the right arm of a man and consist of the subdomains`palm',`thumb' and`¢ngers' [7]. A deep cleft formed by the subdomains is the binding site for the DNA template. In T7 RNAP the dimensions of this cleft allow the placing in it of almost two full turns of dsDNA [9]. Inside this cleft, structural motifs A, B, and C [10] conservative for most single-subunit nucleotide polymerases and containing functionally essential aa residues are located. These residues form the putative active site of T7 RNAP. T7 RNAP catalyzes the transcription from late promoters of bacteriophage T7 recognizing the 2...

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A mutant T7 RNA polymerase (T7 RNAP) having two amino-acid substitutions (Y639F and $641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer. Another T7 RNAP mutant having only the single substitution $641A loses RNA polymerase activity but is able to synthesize DNA.Key words: T7 RNA polymerase; Mutagenesis; dNTP utilization; DNA polymerizing activity distribution of hydroxyl-containing amino-acid residues. Specifically, a serine residue is present at position 641 in T7 RNAP (and at corresponding positions in related RNAPs), while in DNA polymerases (DNAP) no such regularity is observed [5]. As $641 is the hydroxyl-bearing amino-acid residue closest to Y639 we have asked whether the hydroxyl groups of these two residues may be involved in the interactions of enzyme with NTP and, specifically, in discrimination between dNTP and rNTP as potential substrates. To test this, we have generated mutant enzymes with phenylalanine in place of tyrosine at position 639, alanine in place of serine in position 641 and a double mutant bearing both of these substitutions. The substrate specificity and other features of the latter two proteins were found to be quite surprising.