Biosynthesis of RNA polymerase in Escherichia coli

Summary

In an effort to elucidate the control of synthesis of the DNA-dependent RNA polymerase in Escherichia coli, intracellular amounts of the individual subunits were determined by polyacrylamide gel electrophoresis of cell lysates and of precipitates formed with specific antibody against holoenzyme I.

Polyacrylamide gel electrophoresis of cell lysates in the presence of sodium dodecyl sulfate has been believed to separate the two larger subunits, β and β′, of RNA polymerase from the bulk of protein in E. coli. However, a polypeptide unrelated to the polymerase was found to migrate in the immediate vicinity of the β′ subunit, interfering with the accurate measurement of this subunit. Taking account of the presence of this peptide, designated tentatively as χ, the quantity of RNA polymerase was estimated relying only on the β subunit.

Subunits content was also measured by polyacrylamide gel electrophoresis of the precipitates formed by treating cell lysates with anti-holoenzyme I serum. Since the isolated individual subunits as well as the holo- and core enzyme could be precipitated by the antibody, the present procedure permitted to determine total amounts of the subunits within cells.

The subunits α, β and β′, the constituents of core portion of RNA polymerase, were found to be produced coordinately during steady-state growth at different rates within the range examined (0.22 to 1.87 generations/hr) and to be metabolically as stable as the bulk of protein. The rate of synthesis of these subunits relative to the total protein was found to be balanced with the growth rate; the differential rate of synthesis of enzyme core (α_p) can be represented by the following empirical equation: α_p(%) = 0.7 μ + 0.45, where μ represents growth rate (generation/hr). In contrast, the content of σ subunit was considerably small, i.e. only about one third mole equivalent to enzyme core, and was almost unaffected by the rate of growth.