The yeast RET1 gene, which encodes a 130 kd subunit of RNA polymerase III, will be subjected to regional mutagenesis, with subsequent screening for altered RNA polymerase III activity. The RET1 protein belongs to a superfamily that includes the second largest subunit of all nuclear and bacterial polymerases. Common to all family members are 12 protein sequence motifs, in four of which we will bring about amino acid substitutions. Of particular interest is a sequence 77 amino acids in length that is in part homologous to the Rif region of the Beta subunit of E. coli RNA polymerase. Simple amino acid substitutions in many residues throughout the Rif region and the corresponding RET sequences change the response of RNA polymerase to termination signals. By sequencing these RET1 mutations and characterizing the activity of Pol III containing the mutant subunits, we seek to infer the function of this protein region in transcription elongation and termination. Many of the RET1 mutations that block a specific step in transcription are lethal. Pol III with a lethal RET1 mutant protein can only be recovered from heterozygous cells. To obtain from them a pure mutant extract for transcription studies, we have developed an immunoselection method based upon epitope tagging and selective removal of the wild type Pol III.
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