RRN11 encodes the third subunit of the complex containing Rrn6p and Rrn7p that is essential for the initiation of rDNA transcription by yeast RNA polymerase I

• Published in: The Journal of biological chemistry 1996-08, Vol.271 (35), p.21062-21067
• Main Authors: Lalo, D, Steffan, J.S, Dodd, J.A, Nomura, M
• Format: Article
• Language: English
• Subjects: ADN; Amino Acid Sequence; AMINO ACID SEQUENCES; BINDING; BINDING PROTEINS; CHEMICAL COMPOSITION; Cloning, Molecular; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; CORE FACTOR; DNA; DNA, Ribosomal - genetics; DNA-Binding Proteins - metabolism; FACTEUR DE TRANSCRIPTION; FACTORES DE TRANSCRIPCION; Fungal Proteins - genetics; Fungal Proteins - metabolism; GLUTATHIONE TRANSFERASE; Humans; MOLECULAR SEQUENCE DATA; NUCLEOTIDE SEQUENCE; Pol1 Transcription Initiation Complex Proteins; PROTEINAS AGLUTINANTES; PROTEINAS RECOMBINANTES; PROTEINE DE LIAISON; PROTEINE RECOMBINANTE; RECOMBINANT PROTEINS; RIBOSOMAS; RIBOSOME; RIBOSOMES; RNA Polymerase I - metabolism; RRN11 GENE; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; TATA-BINDING PROTEIN; TATA-Box Binding Protein; TRANSCRIPCION; TRANSCRIPTION; TRANSCRIPTION FACTORS; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic; TRANSFERASAS; TRANSFERASE; TRANSFERASES
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.35.21062

A new gene, RRN11, has been defined by certain rrn mutants of Saccharomyces cerevisiae which are defective specifically in the transcription of 35 S rRNA gene by RNA polymerase I (pol I). We have cloned the gene 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 mutant extracts, which were previously shown to be deficient in the essential pol I transcription factor called Rrn6/7 complex or core factor (CF). The CF complex was previously shown to consist of three proteins, the 102- and 60-kDa subunits encoded by RRN6 and RRN7, respectively, and the 66-kDa subunit. The results of the above complementation experiments combined with mobility of Rrn11p in SDS-polyacrylamide gel electrophoresis analysis relative to Rrn6p and Rrn7p led to the conclusion that RRN11 encodes the 66-kDa subunit of CF. Glutathione S-transferase-Rrn11p fusion protein was found to bind strongly to 35S-labeled Rrn6p and Rrn7p but only weakly to 35S-labeled TATA- binding protein. Similarly, glutathione S-transferase-Rrn7p fusion protein bound strongly to 35S-labeled Rrn6p and Rrn11p but only weakly to 35S-labeled TATA-binding protein. These results are consistent with the fact that one can purify CF consisting of Rrn6p, Rrn7p, and Rrn11p from yeast cell extracts, but the purified complex does not contain TATA-binding protein. RRN11 was shown to be an essential gene, and [3H]uridine pulse experiments demonstrated directly that RRN11 is essential for rDNA transcription by pol I in vivo. Thus all three subunits of CF are essential for rDNA transcription. Because of the resemblance of CF to mammalian essential pol I transcription factor SL1, the amino acid sequences of Rrn11p and the other two subunits of CF were compared with those of the three TATA-binding protein-associated factors