Organisms rely on close interplay between DNA replication, recombination, and repair to secure transmission of the genome. In rapidly dividing cells, there is also great pressure for transcription, which may induce conflict with replication. We investigated the potential for conflict in bacterial cells, where there is no temporal separation of these processes. Eliminating the stringent response regulators ppGpp and DksA or the GreA and Mfd proteins, which revive or dislodge stalled transcription complexes, and especially combinations of these factors, is shown to severely reduce viability when DNA repair is also compromised. Both ppGpp and certain RNA polymerase (RNAP) mutations reduce accumulation of backed-up arrays of stalled transcription complexes. We propose these arrays are formidable obstacles to replication that are normally kept in check in wild-type cells by ppGpp, DksA, GreA, and Mfd. When arrays do obstruct replication, the consequences are resolved by one of the many pathways available to rescue stalled forks.

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...

Oligonucleotides of a novel type containing 2P-O-L Lribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter-containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of proteinnucleic acid interactions.z 1999 Federation of European Biochemical Societies.

The mutant T7 RNA polymerase (T7 RNAP), containing two substitutions (Y639F, S641A) was earlier shown to utilize both rNTP and dNTP in a transcription-like reaction. In this report the ability of the enzyme to catalyze DNA primer extension reaction was demonstrated. The efficiency of the reaction essentially depended on the type of the primer sequence, and was significantly higher if the primer coincided with the T7 promoter non-coding sequence. In this case the primer extension reaction proceeded along with de novo RNA synthesis. The length of the product did not exceed 8 nucleotides, indicating that the primer extension reaction proceeds according to the mechanism of the T7 RNAP-catalyzed abortive transcription.z 1998 Federation of European Biochemical Societies.