Specific fluorescent labeling of two functional domains in RNA polymerase alpha subunit.
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 alpha. subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated alpha subunit and alpha 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 alpha 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 alpha dimerization and its binding of beta' subunit. In the isolated alpha subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant alpha-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.
Ozoline
O N
ON
National Institute of Genetics, Mishima, Shizuoka, Japan.
Murakami
K
K
Negishi
T
T
Fujita
N
N
Ishihama
A
A
D003545:Cysteine / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005452:Fluoresceins / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009941:Organomercury Compounds / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic / Q000235:genetics; D014357:Trypsin / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009941:Organomercury Compounds; C001673:fluorescein mercuric acetate; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin; D003545:Cysteine