A study of the quaternary structure of Escherichia coli RNA polymerase using bis(imido esters)

Abstract

The quaternary structure of E. coli RNA polymerase [EC 2.7.7.6] was studied by cross-linking with a periodate-cleavable bis(imido ester), N,N''-bis(2-carboximidoethyl)tartaramide dimethyl ester dihydrochloride (CETD). The cross-linked holoenzyme gives a characteristic 5-band pattern after electrophoresis on sodium dodecyl sulfate[SDS]-polyacrylamide gels. The components of each band were unambiguously identified by MW measurements, comparisons of cross-linking patterns of holoenzyme and core enzyme and periodate cleavage of cross-links followed by a 2nd dimension SDS-polyacrylamide gel electrophoresis. The bands are .alpha..beta. and .alpha..beta.'', .sigma..beta. and .sigma..beta.'', .alpha..sigma..beta. and .alpha..sigma..beta.'', .beta..beta.'', and .sigma..beta..beta.''. Bands 2 and 4 are the most prominent at low reagent concentrations (up to 2.5 mM), but band 5 becomes the most prominent at higher concentrations. There are no bands corresponding to .alpha..alpha. and .alpha..sigma.; a faint band was tentatively identified as .alpha..beta..beta.''. Shorter bis(imido esters) are much less effective cross-linking reagents than CETD, and they do not give rise to any other cross-linked species. On the basis of these observations, a model for the subunit arrangement of RNA polymerase is proposed.