Single crystals of a chimeric T7/T3 RNA polymerase with T3 promoter specificity

Abstract

Two RNA polymerases homologous to bacteriophage T7 RNA polymerase - bacteriophage Sp6 and T3 RNA polymerases - were screened for crystallization under conditions identical or similar to those reported for the growth of large single crystals of T7 RNA polymerase. A number of mutant T7 RNAPs were also screened under these conditions, as were three chimeric RNA polymerases consisting of T7 RNAP N-terminal and T3 RNAP C-terminal sequences. One chimeric polymerase and two mutant polymerases crystallized readily under T7 RNAP crystallization conditions. The chimeric polymerase crystallized in a space group different from T7 RNA polymerase: orthorhombic with unit cell parameters a = 75A˚, b = 98A˚, c = 159A˚; space group P2 12 1 2 1 and 4 molecules per unit cell. This chimeric enzyme exhibits T3 promoter specificity and will make it possible to investigate how structural differences between the T3 and T7 RNA polymerase promoter recognition domains determine their different promoter specificities.