Lill H R, Hartmann G R
Eur J Biochem. 1975 May;54(1):45-53. doi: 10.1111/j.1432-1033.1975.tb04112.x.
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.
缺乏σ亚基的依赖DNA的RNA聚合酶，在0.4M NaCl存在下以单体形式或在低离子强度下以寡聚体形式用基质结合的胰凝乳蛋白酶或胰蛋白酶进行消化。含σ的聚合酶也以同样的方式进行消化。在酶用去污剂解离成亚基以及水解产生的片段后，通过聚丙烯酰胺凝胶电泳跟踪蛋白水解过程。得到以下结果：(a)大的β和β'亚基在单体聚合酶中的切割速率比在寡聚聚合酶中高得多。(b)两个大的亚基以相同的速率被水解。(c)α亚基在聚合酶的单体和寡聚形式中几乎以相同的速率被水解。(d)σ亚基也是如此。(e)这些效应与所用蛋白酶的底物特异性无关。(f)σ亚基对胰凝乳蛋白酶的敏感性比对胰蛋白酶高得多。(g)σ亚基保护大的β和β'亚基不被胰蛋白酶切割。这些结果可以用RNA聚合酶中原聚体-原聚体相互作用的拓扑结构的一个初步模型来解释。根据这个模型，β和β'亚基含有原聚体同源相互作用的两个位点。一个位点可以被σ亚基的附着所阻断。α和σ亚基不直接参与这种结合。
