Crystallisation of DNA-dependent RNA polymerase from Thermus thermophilus HB 8

Abstract

BACTERIAL DNA-dependent RNA polymerase (nucleoside triphosphate–RNA nucleotidyltransferase; EC 2.7.7.6) consists of at least four different subunits (α, β, β′, and σ) and the subunit composition of the holo-and core enzymes are α₂ββ′σ- and α₂ββ′, respectively. These subunits play different roles and the overall reaction catalysed by this enzyme is well organised and highly specific. To understand the mechanism of transcription, a precise knowledge of the structure of this enzyme is indispensable. The structure of RNA polymerase has been studied by small-angle X-ray analysis¹ and electron microscopy^2–6. Data obtained using these methods, however, are not satisfactory, and even contradict each other. X-ray crystallography seems to be^7,8 the most powerful method of analysing the three-dimensional structure of proteins. Since RNA polymerase has not yet been crystallised, however, it has been impossible to apply this technique. We have recently isolated the core enzymes and holoenzymes of RNA polymerase from the thermophilic bacterium, Thermus thermophilus HB 8 (ref. 9). Both enzymes are quite stable not only against heating but also against denaturation by various detergents. One of the reasons why crystallisation of RNA polymerase has not yet been achieved in spite of many publications concerning extensive purification of the enzyme from various sources, is probably the instability of the enzyme. As a first step towards X-ray crystallographic analysis of RNA polymerase, we tried to crystallise the thermophilic core enzymes and holoenzymes in the hope that their extreme stability might help the crystallisation of the enzyme. We have succeeded in crystallising the core enzyme but not the holoenzyme.