Specific fluorescent labeling of two functional domains in RNA polymerase subunit

Olga N. Ozoline, Katsuhiko Murakami, Tomofumi Negishi, Nobuyuki Fujita, Akira Ishihama

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

A monomercury derivative of fluoresceine acetate (FMMA) was previously suggested as a specific reagent reacting with only one of four cysteine (Cys) residues in the α subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated a subunit and α subunit assembled in the holoenzyme. In both cases, the highest reactivity was identified for Cys-269 positioned in the regulatory helix of C-terminal domain (CTD) which includes the contact sites for both class-I transcription factors and DNA UP elements. Substitution of Ala for both Cys-269 and Cys- 176 completely eliminates the reactivity of α subunit against the fluorescent dye, supporting the prediction that another reactive amino acid under native conformation is Cys-176, which is positioned within or near the region important for a dimerization and its binding of β' subunit. In the isolated a subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant α- subunits, bearing only one Cys residue at either 269 or 176, could be reconstituted into locally modified and active enzymes. This FMMA modification system may provide a tool suitable for studies of intra- and intermolecular interactions of this subunit.

Original languageEnglish (US)
Pages (from-to)183-192
Number of pages10
JournalProteins: Structure, Function and Genetics
Volume30
Issue number2
DOIs
StatePublished - Feb 1 1998

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DNA-Directed RNA Polymerases
Labeling
Cysteine
Bearings (structural)
Holoenzymes
Dimerization
Fluorescent Dyes
Escherichia coli
Conformations
Acetates
Substitution reactions
Transcription Factors
Derivatives
Amino Acids
DNA
Enzymes

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Biochemistry
  • Molecular Biology

Cite this

Ozoline, Olga N. ; Murakami, Katsuhiko ; Negishi, Tomofumi ; Fujita, Nobuyuki ; Ishihama, Akira. / Specific fluorescent labeling of two functional domains in RNA polymerase subunit. In: Proteins: Structure, Function and Genetics. 1998 ; Vol. 30, No. 2. pp. 183-192.
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Specific fluorescent labeling of two functional domains in RNA polymerase subunit. / Ozoline, Olga N.; Murakami, Katsuhiko; Negishi, Tomofumi; Fujita, Nobuyuki; Ishihama, Akira.

In: Proteins: Structure, Function and Genetics, Vol. 30, No. 2, 01.02.1998, p. 183-192.

Research output: Contribution to journalArticle

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