Dimeric Association of Escherichia coli RNA Polymerase α Subunits, Studied by Cleavage of Single-Cysteine α Subunits Conjugated to Iron−(S)-1-[p-(Bromoacetamido)benzyl]ethylenediaminetetraacetate

• Published in: Biochemistry (Easton) 1998-02, Vol.37 (5), p.1344-1349
• Main Authors: Miyake, Reiko, Murakami, Katsuhiko, Owens, Jeffrey T, Greiner, Douglas P, Ozoline, Olga N, Ishihama, Akira, Meares, Claude F
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase; Biotinylation; Cysteine - genetics; Cysteine - metabolism; Dimerization; DNA-Directed RNA Polymerases - chemical synthesis; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Edetic Acid - analogs & derivatives; Edetic Acid - metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Hydrolysis; Iron Chelating Agents - metabolism; Models, Molecular; Mutagenesis, Site-Directed; Organometallic Compounds - metabolism; Protein Folding; Streptavidin
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9723313

Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase α subunit were studied using a set of four mutant α subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant α subunits were conjugated with the cutting reagent iron−(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe−BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within ≈12 Å of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.