In vitro thermal inactivation of a temperature-sensitive sigma subunit mutant (rpoD800) of Escherichia coli RNA polymerase proceeds by aggregation.
A temperature-sensitive mutant sigma subunit (rpoD800) purified from Escherichia coli was inactivated in vitro by temperatures in excess of 37 degrees C whereas wild type sigma remained stable up to 49 degrees C. Both temperature-sensitive and wild type sigma formed multimeric aggregates upon thermal inactivation which were visualized by electron microscopy as polymeric chains. Conditions favoring sigma monomer (low sigma concentration and binding to core polymerase) protected temperature-sensitive sigma from heat inactivation. Full activity was recovered from inactivated temperature-sensitive sigma aggregates by incubation in a buffer containing 6 M guanidine HCl and subsequent removal of denaturant by dilution. Both wild type and temperature-sensitive sigma recovered full activity levels, retaining their characteristic thermal inactivation temperatures after denaturation in 6 M guanidine HCl and renaturation. Transcription of T4 DNA by RNA polymerase containing the rpoD800 mutant sigma subunit remained undiminished for 10 min after shift up to 46 degrees C but was almost completely inhibited within the following 10 to 15 min.
D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000201:enzymology*; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009154:Mutation; D009994:Osmolar Concentration; D012234:Rho Factor / Q000235:genetics* / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics*
D046911:Macromolecular Substances; D012234:Rho Factor; D014157:Transcription Factors; D012965:Sodium Chloride; D012321:DNA-Directed RNA Polymerases; D005990:Glycerol