|Budget Amount *help
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Halobacterium halobium, one of the halophilic archaebacteria, grows in extreme halophilic environments. The intracellular concentration of K^+ is also extremely high. The mechanisum of gene transcription in H.halobium and the nature of RNA polymerase involved in this process are still remained to study. We tried to purify the RNA polymerase which requires high salt concentrations for function.
Zilling et al. (1978) identified a high-molecular-weight RNA polymerase from a related H.cutirubrum.
In crude extract of H.halobium cells, we detected two RNA polymerase activities, one requiring high concentrations of salt for maximun activity and the other not requiring it. The high-requiring RNA polymerase was purified up to about 1,000-fold by PEI iteatment, Buty-Toyopearl and glycerol gradient centrifugation. The partially purified RNA polymerase required 3M NaCl for maximum activity DNA-dependent RNA synthesis continued at least until 90 min at 40ﾟC.
The enzyme was resistant to both rifampicin and alpha-amanitin, but was sensitive to lignin, which is a potent inhibitor of influenza virus RNA polymerase. The molecular weight was estimated to be about 45 kDs by glycerol gradient centrifugation. Futher purification of the enzyme and detailed analysis of the enzyme structure are in progress.