A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage.
Bottom Line: Polypeptides of one set are encoded by middle phage genes and are found in the phiKZ virions.Thus, this enzyme is a non-virion phiKZ RNAP responsible for transcription of late phage genes.The phiKZ RNAP lacks identifiable assembly and promoter specificity subunits/factors characteristic for eukaryal, archaeal and bacterial RNAPs and thus provides a unique model for comparative analysis of the mechanism, regulation and evolution of this important class of enzymes.
Affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia Department of Molecular Biology and Biochemistry, Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA.Show MeSH
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Mentions: The phiKZ late promoters are associated with a consensus 5′-TATG-3′ motif, with the last nucleotide being a transcription start site (Figure 4A). To investigate whether this motif is necessary for nvRNAP transcription we systematically replaced each of the four consensus positions of the late phiKZ promoter P119L and tested the resulting mutant templates in in vitro multiple-round run-off transcription assay. In addition, an anticonsensus derivative with all four positions of the motif replaced was tested. Every single substitution at the consensus motif led to dramatic reduction of nvRNAP transcription (Figure 4B, lanes 4–7). In contrast, substitutions outside the consensus, at positions −4 and +2, either had a smaller inhibitory effect or even stimulated nvRNAP transcription (Figure 4B, lanes 3 and 8). No transcription from P119L anticonsensus derivative was detected (Figure 4B, lane 2). We conclude that the late promoter 5′-TATG-3′ conserved motif is necessary for in vitro transcription by nvRNAP.
Affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia Department of Molecular Biology and Biochemistry, Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA.