[12] Genetic dissection of complex heteromultimeric enzymes: The case of yeast DNA-dependent RNA polymerases

Publisher Summary This chapter discusses some genetic methods currently used in Saccharomyces cerevisiae to analyze the organization of complex heteromultimeric yeast enzymes. Based on the studies of yeast DNA-dependent RNA polymerases, the construction of null alleles, the isolation of conditional mutations from mutagenized allele libraries, the characterization of extragenic suppressors, and the use of genetic tests to monitor protein–protein association in vivo are considered in the chapter. It also reviews the properties of yeast RNA polymerases I, II, and III. The enzymes dissociate into 14, 12, and 16 distinct subunits, respectively, after denaturing gel electrophoresis. They transcribe nonspecific DNA matrices in vitro , but depend on a variety of general transcription factors for faithful gene transcription. Almost all of the RNA polymerase genes have now been cloned. In agreement with earlier biochemical and immunological data, sequence comparison has shown that yeast RNA polymerases have a closely related subunit organization. The two large subunits are similar in each of the three enzymes and are related, respectively, to the β' and β subunits of bacterial enzymes.