Physical and functional interaction of the yeast corepressor Tup1 with mRNA 5'-triphosphatase.
The Tup1-Ssn6 complex is an important corepressor in Saccharomyces cerevisiae that inhibits transcription through interactions with the basal transcription machinery and by remodeling chromatin. In a two-hybrid screen for factors that interact with the Schizosaccharomyces pombe Tup1 ortholog, Tup11, we isolated the pct1+ cDNA. The pct1+ gene encodes an mRNA 5'-triphosphatase, which catalyzes the first step of mRNA capping reactions. Pct1 did not interact with the S. pombe Ssn6 ortholog. In vitro glutathione S-transferase pull-down experiments revealed that Pct1 binds to the WD repeat regions of Tup11 and the functionally redundant Tup12 protein. Similarly, the S. cerevisiae Tup1 protein associates with the mRNA 5'-triphosphatase encoded by the CET1 gene. The highly conserved C-terminal domain of Cet1 interacts with Tup1 in vitro, and Tup1-Ssn6 complexes co-purify with the Cet1 protein, indicating that in vivo interactions also occur between these proteins. Over-expression of CET1 compromised repression of an MFA2-lacZ reporter gene that is subject to Tup1-Ssn6 repression. These genetic and biochemical interactions between Tup1-Ssn6 and Cet1 indicate that the capping enzyme associated with RNA polymerase II is a target of the corepressor complex.
Mukai
Yukio
Y
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. mukai@bio.eng.osaka-u.ac.jp
Davie
Judith K
JK
Dent
Sharon Y R
SY
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D015151:Immunoblotting; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
C065455:CYC8 protein, S cerevisiae; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C517520:Ssn6 protein, S pombe; C066502:TUP1 protein, S cerevisiae; C471690:TUP1 protein, S pombe; C471691:Tup11 protein, S pombe; C471692:Tup12 protein, S pombe; C087021:res2 protein, S pombe; D005982:Glutathione Transferase; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase