Direct interactions between the Paf1 complex and a cleavage and polyadenylation factor are revealed by dissociation of Paf1 from RNA polymerase II.
The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3'-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3'-end formation observed in the absence of Paf1.
Nordick
Kristen
K
Department of Biochemistry and Molecular Genetics and Molecular Biology Program, University of Colorado School of Medicine, 12801 East 17th Ave., P.O. Box 6511, Aurora, CO 80045, USA.
Hoffman
Matthew G
MG
Betz
Joan L
JL
Jaehning
Judith A
JA
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000378:metabolism*
C104439:CDC73 protein, S cerevisiae; C104718:CTR9 protein, S cerevisiae; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C097698:PAF1 protein, S cerevisiae; C107273:RTF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035602:Transcriptional Elongation Factors; D039221:mRNA Cleavage and Polyadenylation Factors; D012319:RNA Polymerase II