Formation of a carboxy-terminal domain phosphatase (Fcp1)/TFIIF/RNA polymerase II (pol II) complex in Schizosaccharomyces pombe involves direct interaction between Fcp1 and the Rpb4 subunit of pol II.
In transcriptional regulation, RNA polymerase II (pol II) interacts and forms complexes with a number of protein factors. To isolate and identify the pol II-associated proteins, we constructed a Schizosaccharomyces pombe strain carrying a FLAG tag sequence fused to the rpb3 gene encoding the pol II subunit Rpb3. By immunoaffinity purification with anti-FLAG antibody-resin, a pol II complex containing the Rpb1 subunit with a nonphosphorylated carboxyl-terminal domain (CTD) was isolated. In addition to the pol II subunits, the complex was found to contain three subunits of a transcription factor TFIIF (TFIIF alpha, TFIIF beta, and Tfg3) and TFIIF-interacting CTD-phosphatase Fcp1. The same type of pol II complex could also be purified from an Fcp1-tagged strain. The isolated Fcp1 showed CTD-phosphatase activity in vitro. The fcp1 gene is essential for cell viability. Fcp1 and pol II interacted directly in vitro. Furthermore, by chemical cross-linking, glutathione S-transferase pulldown, and affinity chromatography, the Fcp1-interacting subunit of pol II was identified as Rpb4, which plays regulatory roles in transcription. We also constructed an S. pombe thiamine-dependent rpb4 shut-off system. On repression of rpb4 expression, the cell produced more of the nonphosphorylated form of Rpb1, but the pol II complex isolated with the anti-FLAG antibody contained less Fcp1 and more of the phosphorylated form of Rpb1 with a concomitant reduction in Rpb4. This result indicates the importance of Fcp1-Rpb4 interaction for formation of the Fcp1/TFIIF/pol II complex in vivo.
Kimura
Makoto
M
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. makimura@lab.nig.ac.jp
Suzuki
Hisako
H
Ishihama
Akira
A
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D012319:RNA Polymerase II; C423309:RPB4 protein, S cerevisiae; C499659:rpb4 protein, S pombe; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase; C056604:transcription factor TFIIF