RRN11 ENCODES THE 3RD SUBUNIT OF THE COMPLEX CONTAINING RRN6P AND RRN7P THAT IS ESSENTIAL FOR THE INITIATION OF RDNA TRANSCRIPTION BY YEASTRNA-POLYMERASE-I

A new gene, RRN11, has been defined by certain rrn mutants of Saccharo myces cerevisiae which are defective specifically in the transcription of 35 S rRNA gene by RNA polymerase I (pol I), We have cloned the gen e and found that it encodes a protein of 507 amino acids. We have used a strain with the chromosomal RRN11 deleted and carrying HA1 epitope- tagged RRN11 on a plasmid to isolate a protein complex containing the protein encoded by RRN11, This protein complex complemented rrn6 mutan t extracts, which were previously shown to be deficient in the essenti al pol I transcription factor called Rrn6/7 complex or core factor (CF ), The CF complex was previously shown to consist of three proteins, t he 102- and 60-kDa subunits encoded by RRN6 and RRN7, respectively, an d the 66-kDa subunit. The results of the above complementation experim ents combined with mobility of Rrn11p in SDS-polyacrylamide gel electr ophoresis analysis relative to Rrn6p and Rrn7p led to the conclusion t hat RRN11 encodes the 66-kDa subunit of CF. Glutathione S-transferase- Rrn11p fusion protein was found to bind strongly to S-35-labeled Rrn6p and Rrn7p but only weakly to S-35-labeled TATA-binding protein, Simil arly, glutathione S-transferase-Rrn7p fusion protein bound strongly to S-3S-labeled Rrn6p and Rrn11p but only weakly to S-35-labeled TATA-bi nding protein, These results are consistent with the fact that one can purify CF consisting of Rrn6p, Rrn7p, and Rrn11p from yeast: cell ext racts, but the purified complex does not contain TATA-binding protein, RRN11 was shown to be an essential gene, and [H-3]uridine pulse exper iments demonstrated directly that RRN11 is essential for rDNA transcri ption by pol I in vivo, Thus all three subunits of CF are essential fo r rDNA transcription, Because of the resemblance of CF to mammalian es sential pol I transcription factor SL1, the amino acid sequences of Rr n11p and the other two subunits of CF were compared with those of the three TATA-binding protein-associated factors (TAFs) in the human SL1, TAF(I)48, TAF(I)63, and TAF(I)110. No significant similarity was dete cted between two sets of the proteins. Similarity as well as differenc es between CF and SL1 are discussed.