Specific fluorescent labeling of two functional domains in RNA polymerase α subunit
✍ Scribed by Olga N. Ozoline; Katsuhiko Murakami; Tomofumi Negishi; Nobuyuki Fujita; Akira Ishihama
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 231 KB
- Volume
- 30
- Category
- Article
- ISSN
- 0887-3585
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✦ Synopsis
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 ␣ subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated ␣ subunit and ␣ 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 ␣ 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 ␣ dimerization and its binding of β' subunit. In the isolated ␣ subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant ␣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.