Fluorescent modification of the cysteine 202 residue of Escherichia coli transcription termination factor rho.
The lone cysteine residue (Cys-202) of transcription termination factor rho has been modified with the sulfhydryl-specific dyes 5-iodoacetamidofluorescein and 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid. Labeling with both dyes is specific for the Cys-202 residue and is at least 90% complete. Rho protein is an RNA-dependent ATPase and exists as a hexamer of identical subunits in its activated (RNA-liganded) form. We find that chemical modification of rho at Cys-202 does not significantly change the properties of the protein; subunit assembly, RNA binding, and poly(rC)-activated ATP hydrolysis are all relatively unperturbed by the covalent attachment of these fluorescent moieties. On the other hand, the spectral, quenching, and anisotropy properties of the fluorescent groups are all significantly modified by attachment to the protein. No energy transfer is seen between fluorescein-labeled subunits within rho hexamers, indicating that the Cys-202 residues on these subunits are located at least 40 A apart. These fluorescently labeled rho molecules should represent useful probes to study the conformations and inter- and intrasubunit geometries of this termination factor at various stages of its interaction with nascent RNA transcripts.
Seifried
S E
SE
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Wang
Y
Y
von Hippel
P H
PH
D000251:Adenosine Triphosphatases / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D003545:Cysteine; D004735:Energy Transfer; D004926:Escherichia coli / Q000032:analysis*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D012313:RNA / Q000378:metabolism; D012234:Rho Factor / Q000378:metabolism / Q000494:pharmacology; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents; D014157:Transcription Factors / Q000378:metabolism / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D012234:Rho Factor; D013439:Sulfhydryl Reagents; D014157:Transcription Factors; D012313:RNA; C015296:5-iodoacetamidofluorescein; C006421:1,5-I-AEDANS; D000251:Adenosine Triphosphatases; D003545:Cysteine