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A specific assay for yeast RNA polymerases in crude cell extracts.

With the object of isolating yeast mutants altered in DNA-dependent RNA polymerases, a specific assay for the various classes of RNA polymerases was developed, using crude protein extracts as the source of enzyme. 
 
 
 
Yeast extracts incorporated AMP as well as UMP and CMP residues into acid-precipitable material in template-independent reactions which obscured DNA-dependent RNA polymerase activities. In contrast, GMP incorporation was exclusively template-dependent. Therefore cytidinerich templates were selected. A simple and sensitive assay for RNA polymerase B was based on its ability to use the ribohomopolymer (rC)n as template and its exclusive requirement for Mn2+ as activator cation. The specificity of the assay was further shown by the fact that (rG)n synthesis under these conditions was totally inhibited by 50 μg/ml of α-amanitin, a sensitivity ascribed only to RNA polymerase B. 
 
 
 
The other two forms of enzymes, RNA polymerases A and C, were assayed with d(I-C)n as template in the presence of Mg2+ as activator cation, under the correct ionic conditions. At low ionic strength and in the presence of Mg2+, d(I-C)n-directed synthesis of r(C-G)n was catalyzed by enzymes A and C. In contrast, at high ionic strength or in the presence of 2 mg/ml of α-amanitin, the activity of RNA polymerase A was inhibited (∼ 50%). Thus the activities of A and C enzymes could be calculated by the difference of activities measured on low salt and high salt. It was found that RNA polymerases A and C each contributed to about 50% of the r(G-C)n synthesis. 
 
 
 
Several parameters influencing the assay were investigated. In particular, RNA polymerase activities were found to be independent of cell growth state. In order to use these methods for the rapid screening of a large number of clones, the processing of the assays was modified to permit a rapid comparison by visual tests.