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A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage.

Yakunina M, Artamonova T, Borukhov S, Makarova KS, Severinov K, Minakhin L - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

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.

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Promoter specificity determinants of nvRNAP. (A) Sequence logos of phiKZ middle and late promoters. (B) Analysis of the phiKZ late promoter consensus motif 5′-TATG-3′ by point mutations. Left panel – in vitro run-off transcription by nvRNAP from late promoter P119L and its derivatives. RO – run-off RNA products. Right panel – an alignment of nucleotide sequences of wild-type and mutant promoters (introduced substitutions are shown in bold typeface). The +1 start site is underlined. (C) Mutational analysis of upstream and downstream sequences of phiKZ late promoter P119L. Left panel – in vitro run-off transcription by nvRNAP from chimeric templates based on phiKZ P78M middle and P119L late promoters. RO – run-off RNA products. Right panel – schematic representation of hybrid promoters used. The P119L sequence is shown by a thin line; the P78M is shown by a thick line. Numbers above the scheme indicate upstream (position −53 with respect to the start site) and downstream (position +51) boundaries of promoter fragments used. The late consensus TATG motif with the +1 start of transcription is underlined. The putative middle promoter conserved motif 5′-AAAATTACC-3′ is also indicated. The numbers at the right indicate the transcription activities relative to the positive control (wild-type P119L). Average values and standard deviations from three independent experiments are presented.
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Figure 4: Promoter specificity determinants of nvRNAP. (A) Sequence logos of phiKZ middle and late promoters. (B) Analysis of the phiKZ late promoter consensus motif 5′-TATG-3′ by point mutations. Left panel – in vitro run-off transcription by nvRNAP from late promoter P119L and its derivatives. RO – run-off RNA products. Right panel – an alignment of nucleotide sequences of wild-type and mutant promoters (introduced substitutions are shown in bold typeface). The +1 start site is underlined. (C) Mutational analysis of upstream and downstream sequences of phiKZ late promoter P119L. Left panel – in vitro run-off transcription by nvRNAP from chimeric templates based on phiKZ P78M middle and P119L late promoters. RO – run-off RNA products. Right panel – schematic representation of hybrid promoters used. The P119L sequence is shown by a thin line; the P78M is shown by a thick line. Numbers above the scheme indicate upstream (position −53 with respect to the start site) and downstream (position +51) boundaries of promoter fragments used. The late consensus TATG motif with the +1 start of transcription is underlined. The putative middle promoter conserved motif 5′-AAAATTACC-3′ is also indicated. The numbers at the right indicate the transcription activities relative to the positive control (wild-type P119L). Average values and standard deviations from three independent experiments are presented.

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.


A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage.

Yakunina M, Artamonova T, Borukhov S, Makarova KS, Severinov K, Minakhin L - Nucleic Acids Res. (2015)

Promoter specificity determinants of nvRNAP. (A) Sequence logos of phiKZ middle and late promoters. (B) Analysis of the phiKZ late promoter consensus motif 5′-TATG-3′ by point mutations. Left panel – in vitro run-off transcription by nvRNAP from late promoter P119L and its derivatives. RO – run-off RNA products. Right panel – an alignment of nucleotide sequences of wild-type and mutant promoters (introduced substitutions are shown in bold typeface). The +1 start site is underlined. (C) Mutational analysis of upstream and downstream sequences of phiKZ late promoter P119L. Left panel – in vitro run-off transcription by nvRNAP from chimeric templates based on phiKZ P78M middle and P119L late promoters. RO – run-off RNA products. Right panel – schematic representation of hybrid promoters used. The P119L sequence is shown by a thin line; the P78M is shown by a thick line. Numbers above the scheme indicate upstream (position −53 with respect to the start site) and downstream (position +51) boundaries of promoter fragments used. The late consensus TATG motif with the +1 start of transcription is underlined. The putative middle promoter conserved motif 5′-AAAATTACC-3′ is also indicated. The numbers at the right indicate the transcription activities relative to the positive control (wild-type P119L). Average values and standard deviations from three independent experiments are presented.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Promoter specificity determinants of nvRNAP. (A) Sequence logos of phiKZ middle and late promoters. (B) Analysis of the phiKZ late promoter consensus motif 5′-TATG-3′ by point mutations. Left panel – in vitro run-off transcription by nvRNAP from late promoter P119L and its derivatives. RO – run-off RNA products. Right panel – an alignment of nucleotide sequences of wild-type and mutant promoters (introduced substitutions are shown in bold typeface). The +1 start site is underlined. (C) Mutational analysis of upstream and downstream sequences of phiKZ late promoter P119L. Left panel – in vitro run-off transcription by nvRNAP from chimeric templates based on phiKZ P78M middle and P119L late promoters. RO – run-off RNA products. Right panel – schematic representation of hybrid promoters used. The P119L sequence is shown by a thin line; the P78M is shown by a thick line. Numbers above the scheme indicate upstream (position −53 with respect to the start site) and downstream (position +51) boundaries of promoter fragments used. The late consensus TATG motif with the +1 start of transcription is underlined. The putative middle promoter conserved motif 5′-AAAATTACC-3′ is also indicated. The numbers at the right indicate the transcription activities relative to the positive control (wild-type P119L). Average values and standard deviations from three independent experiments are presented.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus