ISSN:
0887-3585
Keywords:
chemical modification
;
fluorescent probe
;
site-directed mutagenesis
;
cysteine-free protein
;
alanine scanning
;
enzyme reconstitution
;
protein-DNA interaction
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
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 α 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 α dimerization and its binding of β' subunit. In the isolated α 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. Proteins 30:183-192, 1998. © 1998 Wiley-Liss, Inc.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
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