A. DEPARTMENT OF MOLECULAR GENETICS
A-c. Molecular Mechanism Laboratory- Hiroshi Mitsuzawa

RESEARCH ACTIVITIES

(1) Identification of glyceraldehyde-3-phosphate dehydrogenase and actin as proteins that interact with the Rpb7 subunit of RNA polymerase II

--Eukaryotic RNA polymerase II (pol II), the enzyme that synthesizes all mRNAs and some non-coding RNAs, plays a central role in transcription. pol II consists of evolutionarily conserved 12 subunits, designated Rpb1 to Rpb12. The catalytic activity resides in a 10-subunit core, whereas the initiation of transcription requires two additional subunits, Rpb4 and Rpb7, which form a heterodimer to associate with the core.
--To gain insight into the role of the Rpb7 subunit, we performed a two-hybrid screen by using the fission yeast Schizosaccharomyces pombe Rpb7 protein as bait. The screen yielded glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. GAPDH and actin were also found to be associated with S. pombe pol II complexes. Moreover, GAPDH and actin were affinity-purified from S. pombe extract with an Rpb4/Rpb7-coupled column. Although GAPDH is a well-characterized glycolytic enzyme abundant in the cytoplasm, nuclear GAPDH in human cells has been identified as a key component of a coactivator complex for histone H2B transcription. In addition, it has recently been reported that actin is required for formation of the preinitiation complex. Our results are consistent with these observations and further suggest that GAPDH and actin play roles in pol II transcription through the interaction with Rpb7.

(2) Identification of the general transcription factor TFIID in S. pombe

Hiroshi Mitsuzawa

--TFIID, one of the general transcription factors required for transcription initiation by pol II, comprises the TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs). Unlike those in human, Drosophila, and Saccharomyces cerevisiae, TAFs in S. pombe have not been characterized until recently. We have identified five S. pombe TAFs biochemically: spTAF111/130 (spTAF1), spTAF72 (spTAF5), spTAF73 (spTAF5b), spTAF50 (spTAF6), and Ptr6 (spTAF7).^{1, 2)} (Given in parentheses are new unified names proposed recently.) Of particular interest are spTAF72 and spTAF73, which are likely to regulate genes involved in progression through M phase of the cell cycle. spTAF50 was shown to interact with the WD40-repeat domain of spTAF72. These interacting TAFs were found to be shared by the TFIID and SAGA complexes. Furthermore, we carried out BLAST searches for putative TAFs, revealing that the S. pombe genome contains nearly all TAF genes identified in other organisms.^{1, 2)}

PUBLICATIONS

Reviews
1. Mitsuzawa, H. and Ishihama, A. (2004). RNA polymerase II transcription apparatus in Schizosaccharomyces pombe. Curr. Genet. 44, 287-294.

Books
2. Kimura, M., Mitsuzawa, H. and Ishihama, A. (2004). RNA polymerases and accessory factors. In The Molecular Biology of Schizosaccharomyces pombe. R. Egel, ed. (Heidelberg, Germany: Springer-Verlag), pp.329-342.