Thiol Function and Tertiary Structure of RNA Polymerase of Escherichia coli

Authors


Abstract

  • 1RNA polymerase requires thiol compounds to maintain optimum activity during isolation, although it is not a thiol enzyme.
  • 2Total thiols have been determined for RNA core polymerase by reaction with p-chloro-mercuribenzoate, 2-chloromercuri-4-nitrophenol and 5,5′-dithio-bis(2-nitrobenzoic acid). Their distribution between the subunits has been determined as ααββ' = 4.4.8.8.
  • 3An arbitrary division of the reaction rate profile shows that of the 18 thiols reacting with 5,5′-dithio-bis(2-nitrobenzoec acid) in 18 h, over 10 react in 1% of this time. Most of these are buried thiols since only 2 or 3 react with N-[p-(-methyl-2-benzthiazole)phenyl]maleimide.
  • 4Enzyme activity is lost gradually on titration with 5,5′-dithio-bis(2-nitrobenzoic acid), but rapidly with p-chloromercuribenzoate until 10–12 thiols have reacted per mole; polymerase then undergoes a large reversible change in activity, although with 5,5′-dithiobis(2-nitrobenzoic acid) reversibility is slow and depends on the groups/mole that have reacted.
  • 5This inactivation by 5,5′-dithio-bis(2-nitrobenzoic acid) may be prevented by pre-initiating the enzyme which causes the reaction to stop at 11 thiols/mole, leaving 60–70% of the original activity. DNA on its own does not produce this resistance, which is proportional to the amount of initiated complex present.
  • 6Measurements with fluorescent probes show that considerable destabilisation of the tertiary structure occurs on reaction with p-chloromercuribenzoate. Consideration of this together with the known subunit distribution of thiols strongly suggests that DNA acts by stabilising the structure rather than by direct steric hindrance.
Enzyme
 

RNA polymerase or nucleoside triphosphate: RNA nucleotidyltransferase (DNA dependent) (EC 2.7.7.6)

Ancillary