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Molecular and Cellular Biology, March 2002, p. 1577-1588, Vol. 22, No. 5
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.5.1577-1588.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Makoto Kimura,1* Hisako Suzuki,1,2 and Akira Ishihama1

Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540 ,1 Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan2

Received 15 October 2001/ Returned for modification 13 November 2001/ Accepted 30 November 2001

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}, TFIIFß, 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.

* Corresponding author. Mailing address: Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. Phone: 81-559-81-6742. Fax: 81-559-81-6746. E-mail: makimura{at}lab.nig.ac.jp.

Molecular and Cellular Biology, March 2002, p. 1577-1588, Vol. 22, No. 5
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.5.1577-1588.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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