Quantitation of RNA polymerase subunits in Escherichia coli during exponential growth and after bacteriophage T4 infection

Summary

A method was developed to measure the amounts of RNA polymerase subunits, α, β, β′ and σ in crude extracts of Escherichia coli. The proteins were labelled by growing the cells in ³⁵S-sulphate containing media. For measuring β and β′, the cell lysate was electrophoresed on 6% polyacrylamide gels containing SDS and the β and β′ bands cut out and counted. For measuring α and σ, the cell lysate was co-electrophoresed with dansylated RNA polymerase on 8% polyacrylamide gels containing SDS. The fluorescent bands were cut out, the proteins eluted, and the α and σ subunits further purified on polyacrylamide gels containing 8 molar urea.

The results are: (1) β′ is the subunit of the core RNA polymerase which is present in limiting amount. (2) The core enzyme, as measured by β′, constitutes a constant fraction of total cellular protein (0.9%), independent of the bacterial growth rate. (3) The α subunit is made in excess and is probably regulated independently. (4) The σ subunit is present in 0.3–0.4 times the amount of the core enzyme. (5) All four subunits are fully conserved after bacteriophage T4 infection.