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Mutational analysis of the role of the first helix of region 4.2 of the σ70 subunit ofEscherichia coli RNA polymerase in transcriptional activation by activator protein PhoB

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Abstract

Transcription of the genes belonging to the phosphate (pho) regulon inEscherichia coli requires the specific activator protein PhoB, in addition to RNA polymerase containing the major sigma factor, σ70, which is encoded byrpoD. We previously isolated two mutant σ70s(D570G and E575K) that were specifically defective in transcribing thepho genes. The mutated sites were located near and within the first helix of the helix-turn-helix (HTH) motif of region 4.2 of σ70. To study further the role of the first helix of the HTH motif of σ70 in transcriptional activation by PhoB, we made a series ofrpoD mutations that alter the motif and purified the mutant σ70 proteins. RNA polymerases containing the mutant σ70s Y571A, T572L, V576T, K578E and F580V showed reduced in vitro transcription from thepstS promoter, a representativepho promoter, in the presence of PhoB, whereas RNA polymerase containing another mutant σ70 (E574 K) showed enhanced transcription from the promoter. Transcription from the activator-independenttac promoter and thepBR-P4 promoter, which is independent of PhoB and requires cAMP-CRP (cAMP receptor protein) for transcription, was affected at most only marginally by these σ70 mutations. These results provide further evidence that the first helix plays an important role in the specific interaction between RNA polymerase and PhoB protein bound to thepho promoters in transcriptional activation.

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Author notes

  1. S. -K. Kim

    Present address: Department of Biological Sciences, Purdue University, 47907, West Lafayette, IN, USA

  2. A. Nakata

    Present address: Faculty of Pharmacology, Fukuyama University, 1 Gakuen-cho, Fukuyama, 729-03, Hiroshima, Japan

Authors and Affiliations

  1. Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 565, Osaka, Japan

    S. -K. Kim, K. Makino, M. Amemura & A. Nakata

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  1. S. -K. Kim
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  2. K. Makino
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  3. M. Amemura
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  4. A. Nakata
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  5. H. Shinagawa
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Communicated by M. Sekiguchi

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Kim, S.K., Makino, K., Amemura, M. et al. Mutational analysis of the role of the first helix of region 4.2 of the σ70 subunit ofEscherichia coli RNA polymerase in transcriptional activation by activator protein PhoB. Molec. Gen. Genet. 248, 1–8 (1995). https://doi.org/10.1007/BF02456607

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  • DOI: https://doi.org/10.1007/BF02456607

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