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T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis

Arbeitsgruppe Molekulare Genetik, Fakultät für Biologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany¹
Institute of Biochemistry, Laboratory of Gene Engineering, Vilnius 2600, Lithuania²

Author for correspondence: Wolfgang Rüger. Tel: +49 234 32 23102. Fax: +49 234 32 14195. e-mail: wolfgang.rueger{at}ruhr-uni-bochum.de

The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli ⁷⁰ promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase. The -10, -16, -42 and -52 regions are important for transcript initiation with the native polymerase. To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the -10, -16 and -35 regions, but also that of position -33, and even more importantly, maintenance of the upstream promoter element at position -42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters.

Keywords: ADP-ribosylation, modified RNA polymerase, T4 phage, prokaryotic promoter, mutation analysis

Abbreviations: Alt, T4 ADP-ribosyltransferase; PU, promoter unit

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