Mapping protein-protein interactions with a library of tethered cutting reagents: The binding site of σ70 on Escherichia coli RNA polymerase

Stacey L. Traviglia, Saul A. Datwyler, Claude F. Meares

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

Surface-exposed lysine amino groups and other reactive nucleophiles of the σ70 protein were conjugated with the cutting reagent iron (S)-1-[p- (bromoacetamido)benzyl]ethylenediaminetetraacetate (FeBABE) via 2- iminothiolane (2IT) with low efficiency. The result is a library of σ70 conjugates, with an average of 1-2 cutting reagents tethered to any of a variety of sites (lysine, cysteine, etc.) on the surface of the protein. Model calculations indicate that the conjugates in this library should be capable of cutting nearby sites on the backbone of almost any protein or nucleic acid to which σ70 binds. Since cutting occurs only when the protein is bound, the cleaved sites indicate proximity; since only proximal sites are cleaved, interpretation of the results is straightforward. We used this library to map the periphery of the binding site on the core enzyme (α2ββ') of Escherichia coli RNA polymerase. The β subunit was cut primarily within its conserved regions C, D, Rif I, and G; additional sites were also cut between A and B and near conserved regions E and H. The cut sites within the β' subunit were intensely clustered between residues 250- 450, which include its conserved regions C and D, along with two additional cut sites in conserved regions A and G. No cut sites on the α subunit were observed. These results recapitulate and extend those obtained using FeBABE conjugates of seven strategically placed single-Cys σ70 mutants [Owens, J. T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A., and Meares, C. F. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6021-6026]. This technique provides a straightforward, general approach to mapping protein interactions without mutagenesis.

Original languageEnglish (US)
Pages (from-to)4259-4265
Number of pages7
JournalBiochemistry
Volume38
Issue number14
DOIs
StatePublished - Apr 6 1999

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Mapping protein-protein interactions with a library of tethered cutting reagents: The binding site of σ<sup>70</sup> on Escherichia coli RNA polymerase'. Together they form a unique fingerprint.

  • Cite this