RRN6 AND RRN7 ENCODE SUBUNITS OF A MULTIPROTEIN COMPLEX ESSENTIAL FORTHE INITIATION OF RDNA TRANSCRIPTION BY RNA-POLYMERASE-I IN SACCHAROMYCES-CEREVISIAE

Previously, we have isolated mutants of Saccharomyces cerevisiae prima rily defective in the transcription of 358 rRNA genes by RNA polymeras e I and have identified a number of genes (RRN genes) involved in this process. We have now cloned the RRN6 and RRN7 genes, determined their nucleotide sequences, and found that they encode proteins of calculat ed molecular weights of 102,000 and 60,300, respectively. Extracts pre pared from rrn6 and rrn7 mutants were defective in in vitro transcript ion of rDNA templates. We used extracts from strains containing epitop e-tagged wild-type Rrn6 or Rrn7 proteins to purify protein components that could complement these mutant extracts. By use of immunoaffinity purification combined with biochemical fractionation, we obtained a hi ghly purified preparation (Rrn6/7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparent molecular weight of 66,000 , but which did not contain the TATA-binding protein (TBP). This compl ex complemented both rrn6 and rrn7 mutant extracts. Template commitmen t experiments carried out with this purified Rrn6/7 complex and with r rn6 mutant extracts have demonstrated that the Rrn6/7 complex does not bind stably to the rDNA template by itself, but its binding is depend ent on the initial binding of some other factor(s) and that the Rrn6/7 complex is required for the formation of a transcription-competent pr einitiation complex. These observations are discussed in comparison to in vitro rDNA transcription systems from higher eukaryotes.