RRN6 and RRN7 encode subunits of a multiprotein complex essential for the initiation of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae.
Previously, we have isolated mutants of Saccharomyces cerevisiae primarily defective in the transcription of 35S rRNA genes by RNA polymerase 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 calculated molecular weights of 102,000 and 60,300, respectively. Extracts prepared from rrn6 and rrn7 mutants were defective in in vitro transcription of rDNA templates. We used extracts from strains containing epitope-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 highly 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 complex complemented both rrn6 and rrn7 mutant extracts. Template commitment experiments carried out with this purified Rrn6/7 complex and with rrn6 mutant extracts have demonstrated that the Rrn6/7 complex does not bind stably to the rDNA template by itself, but its binding is dependent on the initial binding of some other factor(s) and that the Rrn6/7 complex is required for the formation of a transcription-competent preinitiation complex. These observations are discussed in comparison to in vitro rDNA transcription systems from higher eukaryotes.
Keys
D A
DA
Department of Biological Chemistry, University of California, Irvine 92717-1700.
Vu
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L
Steffan
J S
JS
Dodd
J A
JA
Yamamoto
R T
RT
Nogi
Y
Y
Nomura
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D035463:Pol1 Transcription Initiation Complex Proteins; D012318:RNA Polymerase I / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D005656:Fungal Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; C090311:RRN6 protein, S cerevisiae; C090315:RRN7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012318:RNA Polymerase I