Analysis of regulation mechanisms of the fission yeast RNA polymerase II

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The most remarkable regulation mechanism of RNA polymerase II (Pol II) is reversible phosphorylation of the C-terminal domain (CTD) of Rpb1 subunit during a transcription cycle. A complex of CTD-phosphatase Fcp1/general transcription factor TFIIF/Pol II, which also contains Tfg3, was isolated from Schzosaccharomyces pombe by the head investigator and coworkers previously, and the Rpb4 subunit was identified as the Fcp1 binding site on Pol II. In this project, the following results were obtained.1.Characterization of Tfg3 revealed the following.(1)S.pombe Tfg3 is a TFIIF subunit.(2)Tfg3 is a subunit of TFIID as well, and the Tfg3 association to TFIID is temperature dependent in vivo and in vitro.(3)S.pombe tfg3^- strains are sensitive to a variety of stresses.(4)Multi copy suppressor genes of the tfg3^- mutation were isolated.(5)Tfg3 functions in a gene specific manner at higher temperature.2.Structure-function analysis of Rpb4 subunit of Pol II revealed the following.(1)The 2nd and 3rd of 7 predicted helices of Rpb4 are essential for the Rpb4 assembly to Pol II.(2)Deletion of the 1st helix caused thermo-sensitivity to the cell and CTD hyper-phosphorylation to the unengaged Pol II complex, suggesting a regulatory role of Rpb4 on Fcp1.(3)Deletion of the 4th to 7th helices also caused thermo-sensitivity to the cell. In the cell, more Fcp1 binds to engaged Pol II complex at permissive temperature and the CTD becomes hypo-phosphorylated.3.The following proteins were identified biochemically as Rpb4/Rpb7 binding proteins and novel regulation mechanisms of Pol II were suggested.(1)Glyceraldehyde-3-phosphate dehydrogenase (reported as a component of mammalian transcription mediator complex).(2)Actin (reported as a component of chromatin remodeling factor complex).