Digestion with matrix-bound proteases as a possible probe for the topography of the DNA-dependent RNA polymerase from Escherichia coli.
DNA-dependent RNA polymerase lacking subunit sigma was digested with matrix-bound chymotrypsin or trypsin in the presence of 0.4 M NaCl in the monomeric form or at low ionic strength in the oligomeric form. Sigma-containing polymerase was digested in the same way. The course of proteolysis was followed by polyacrylamide gel electrophoresis after dissociation of the enzyme with detergent into subunits and the fragments produced by the hydrolysis. The following results were obtained. (a) The large subunits beta and beta' are cleaved with a much higher rate in the monomeric than in the oligomeric polymerase. (b) Both large subunits are hydrolysed with the same rate. (c) Subunit alpha is hydrolysed almost with the same rate in the monomeric and oligomeric form of polymerase. (d) The same was found for subunit sigma. (e) These effects were independent of the substrate specificity of the protease used. (f) Subunit sigma is much more susceptible to chymotrypsin than to trypsin. (g) Subunit sigma protects the large subunits beta and beta' against tryptic cleavage. These results can be explained in terms of a tentative model for the topography of the protomer-protomer interactions in RNA polymerase. According to this model subunits beta and beta' contain two sites for isologous interactions of protomers. One site can be blocked by attachment of subunit sigma. Subunits alpha and sigma do not participate directly in the association.
D000178:Acrylamides; D001665:Binding Sites; D002918:Chymotrypsin; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008298:Maleates; D009994:Osmolar Concentration; D011108:Polymers; D011487:Protein Conformation; D012965:Sodium Chloride; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin
D000178:Acrylamides; D046911:Macromolecular Substances; D008298:Maleates; D011108:Polymers; D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D002918:Chymotrypsin; D014357:Trypsin