SPECIFIC FLUORESCENT LABELING OF 2 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 cystei ne (Cys) residues in the alpha subunit of Escherichia coli RNA polymer ase. Here, we analyzed the reactivity against FMMA of both isolated al pha subunit and alpha subunit assembled in the holoenzyme. In both cas es, the highest reactivity was identified for Cys-269 positioned in th e regulatory helix of C-terminal domain (CTD) which includes the conta ct sites for both class-I transcription factors and DNA UP elements. S ubstitution of Ala for both Cys-269 and Cys-176 completely eliminates the reactivity of alpha subunit against the fluorescent dye, supportin g the prediction that another reactive amino acid under native conform ation is Cys-176, which is positioned within or near the region import ant for or dimerization and its binding of beta' subunit. In the isola ted alpha subunit, the reactivity against FMMA is different between th ese 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, cou ld be reconstituted into locally modified and active enzymes. This FMM A modification system may provide a tool suitable for studies of intra - and intermolecular interactions of this subunit. (C) 1998 Wiley-Liss , Inc.