Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease

J. T. Owens, R. Miyake, K. Murakami, A. J. Chmura, N. Fujita, A. Ishihama, C. F. Meares

Research output: Contribution to journalArticle

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Abstract

The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several σ subunits. To characterize the proximity between σ70, the major σ for transcription of the growth-related genes, and the core enzyme subunits (α2ββ'), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(p- bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of σ70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE- conjugated σ70 was bound to the core enzyme, which led to cleavage of nearby sites on the β and β' subunits (but not α). Unlike the results of random cleavage [Greiner, D. P., Hughes, K. A., Gunasekera, A. H. and Meares, C. F. (1996) Proc. Natl. Acad. Sci. USA 93, 71-75], the cut sites from different probe-modified σ70 proteins are clustered in distinct regions of the subunits. On the β subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the β' subunit, the cleavage was identified within the sequence 228-461, including β' conserved regions C and D (which comprise part of the catalytic center).

Original languageEnglish (US)
Pages (from-to)6021-6026
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume95
Issue number11
DOIs
StatePublished - May 26 1998

Fingerprint

DNA-Directed RNA Polymerases
Peptide Hydrolases
Escherichia coli
Enzymes
Catalytic RNA
Edetic Acid
Cysteine
Proteins
Growth
Genes
1-(4-bromoacetamidobenzyl)EDTA

Keywords

  • FeBABE
  • Protein cleavage
  • Single cysteine mutants
  • Tethered FeEDTA

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease. / Owens, J. T.; Miyake, R.; Murakami, K.; Chmura, A. J.; Fujita, N.; Ishihama, A.; Meares, C. F.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 95, No. 11, 26.05.1998, p. 6021-6026.

Research output: Contribution to journalArticle

Owens, J. T. ; Miyake, R. ; Murakami, K. ; Chmura, A. J. ; Fujita, N. ; Ishihama, A. ; Meares, C. F. / Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease. In: Proceedings of the National Academy of Sciences of the United States of America. 1998 ; Vol. 95, No. 11. pp. 6021-6026.
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