Mode of binding of RNA polymerase α subunit to the phased A-tracts upstream of the phospholipase C gene promoter of Clostridium perfringens

Seiichi Katayama, Kotaro Ishibashi, Kazuyoshi Goto, Daisuke Nakamura

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Three phased A5-6-tracts lie upstream of the promoter of plc encoding the α-toxin (phospholipase C) of Clostridium perfringens. The α subunits of C.perfringens RNA polymerase bind directly to the phased A-tracts via the C-terminal domain of the α subunit (αCTD). To identify the amino acid residues involved in the binding of C.perfringens α subunits to the phased A-tracts, 27 amino acid residues in C.perfringens αCTD were substituted with alanine. The affinities of the mutated α subunits for the phased A-tracts were examined by gel shift assays and surface plasmon resonance (SPR). The SPR analyses revealed that the phased A-tracts themselves facilitated a complex formation between the phased A-tracts and C.perfringens α subunits [Kd was 6.1 (±0.3)×10-8M], and that Arg261, Asn264, Gly292 and Lys294 in C.perfringens αCTD were critical for the binding to the phased A-tracts. The topology of these amino acid residues on the predicted structure of C.perfringens αCTD indicated a contact path with the phased A-tracts that was similar to that of Escherichia coli αCTD with the upstream (UP) element. On the other hand, SPR analyses at different temperatures (15, 25 and 37°C) indicated that the affinity of the C.perfringens α subunits for the phased A-tracts increased in a low-temperature-dependent manner, whereas that of the E.coli α subunit for the UP element did not. This suggests that the phased A-tracts may not simply be a subset of the UP element, and that they show specific binding activity with the RNA polymerase α subunit.

Original languageEnglish
Pages (from-to)62-69
Number of pages8
JournalAnaerobe
Volume23
DOIs
Publication statusPublished - Oct 2013
Externally publishedYes

Fingerprint

Clostridium perfringens
Surface Plasmon Resonance
Type C Phospholipases
DNA-Directed RNA Polymerases
Amino Acids
Escherichia coli
Genes
RNA Polymerase III
Temperature
Alanine
Gels

Keywords

  • α subunit of RNA polymerase
  • α-toxin (phospholipase C)
  • Clostridium perfringens
  • Phased A-tracts
  • Surface plasmon resonance (SPR)
  • UP element

ASJC Scopus subject areas

  • Microbiology
  • Infectious Diseases

Cite this

Mode of binding of RNA polymerase α subunit to the phased A-tracts upstream of the phospholipase C gene promoter of Clostridium perfringens. / Katayama, Seiichi; Ishibashi, Kotaro; Goto, Kazuyoshi; Nakamura, Daisuke.

In: Anaerobe, Vol. 23, 10.2013, p. 62-69.

Research output: Contribution to journalArticle

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abstract = "Three phased A5-6-tracts lie upstream of the promoter of plc encoding the α-toxin (phospholipase C) of Clostridium perfringens. The α subunits of C.perfringens RNA polymerase bind directly to the phased A-tracts via the C-terminal domain of the α subunit (αCTD). To identify the amino acid residues involved in the binding of C.perfringens α subunits to the phased A-tracts, 27 amino acid residues in C.perfringens αCTD were substituted with alanine. The affinities of the mutated α subunits for the phased A-tracts were examined by gel shift assays and surface plasmon resonance (SPR). The SPR analyses revealed that the phased A-tracts themselves facilitated a complex formation between the phased A-tracts and C.perfringens α subunits [Kd was 6.1 (±0.3)×10-8M], and that Arg261, Asn264, Gly292 and Lys294 in C.perfringens αCTD were critical for the binding to the phased A-tracts. The topology of these amino acid residues on the predicted structure of C.perfringens αCTD indicated a contact path with the phased A-tracts that was similar to that of Escherichia coli αCTD with the upstream (UP) element. On the other hand, SPR analyses at different temperatures (15, 25 and 37°C) indicated that the affinity of the C.perfringens α subunits for the phased A-tracts increased in a low-temperature-dependent manner, whereas that of the E.coli α subunit for the UP element did not. This suggests that the phased A-tracts may not simply be a subset of the UP element, and that they show specific binding activity with the RNA polymerase α subunit.",
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