HETEROGENEITY OF ESCHERICHIA-COLI RNA-POLYMERASE REVEALED BY HIGH-PRESSURE

The activity and subunit association of Escherichia coli RNA polymerase has been investigated by high pressure techniques (up to 2000 atm). The extent of subunit dissociation in the presence and absence of DNA was monitored by carrying out electrophoresis directly at elevated pressure. The degree of inactivation brought about by high pressure was determined by measuring the enzyme activity following decompression. The loss of activity if the enzyme molecules are not actively involved in transcription is correlated with the extent of association of the polymerase subunits. At any particular pressure only a fraction of polymerase molecules becomes inactivated; the remaining fraction retains its original activity characteristics. If the enzyme molecules are actively involved in transcription when the high pressure is applied, the RNA synthesis can be completely halted, but the elongation activity is fully recovered on decompression. The experimental results are consistent with the existence of a broad distribution of protein conformers that are differentially sensitive to the level of pressure. RNA polymerase molecules that display similar catalytic properties have large differences in their free energy of subunit association in the absence of substrates. (C) 1995 Academic Press Limited