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

The activity and subunit association of Escherichia coli RNA polymeras e 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 pre ssure. The degree of inactivation brought about by high pressure was d etermined by measuring the enzyme activity following decompression. Th e loss of activity if the enzyme molecules are not actively involved i n transcription is correlated with the extent of association of the po lymerase subunits. At any particular pressure only a fraction of polym erase molecules becomes inactivated; the remaining fraction retains it s original activity characteristics. If the enzyme molecules are activ ely involved in transcription when the high pressure is applied, the R NA synthesis can be completely halted, but the elongation activity is fully recovered on decompression. The experimental results are consist ent with the existence of a broad distribution of protein conformers t hat are differentially sensitive to the level of pressure. RNA polymer ase molecules that display similar catalytic properties have large dif ferences in their free energy of subunit association in the absence of substrates. (C) 1995 Academic Press Limited