Abstract
The multiprotein Mediator complex is a coactivator required for transcriptional activation of RNA polymerase II transcribed genes by DNA binding transcription factors. We previously partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis of proteins present in the most highly enriched Mediator fractions by tandem mass spectrometry led to the identification of several new mammalian Mediator subunits, as well as several potential Mediator subunits. Here we identify one of these proteins, encoded by the previously uncharacterized AK000411 open reading frame, as a new subunit of the mammalian Mediator complex. The AK000411 protein, which we designate hIntersex (human Intersex), shares significant sequence similarity with the Drosophila melanogaster intersex protein, which has functional properties expected of a transcriptional coactivator specific for the Drosophila doublesex transactivator. In addition, we show that hIntersex assembles into a subcomplex with Mediator subunits p28b and TRFP. Taken together, our findings identify a new subunit of the mammalian Mediator and shed new light on the architecture of the mammalian Mediator complex.
Highlights
From the ‡Stowers Institute for Medical Research, Kansas City, Missouri 64110, the ¶Division of Biology, California Institute of Technology, Pasadena, California 91125, the ʈDepartment of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160, and the **Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
Analysis of proteins present in the most highly enriched Mediator fractions by tandem mass spectrometry led to the identification of several new mammalian Mediator subunits, as well as several potential Mediator subunits
Analysis of proteins present in our most highly enriched Mediator fractions by tandem mass spectrometry led to the identification of a large number of the known mammalian Mediator subunits, as well as a collection of proteins not previously recognized as subunits of the mammalian Mediator complex
Summary
We identify one of these proteins, encoded by the previously uncharacterized AK000411 open reading frame, as a new subunit of the mammalian Mediator complex. Analysis of proteins present in the most highly enriched Mediator fractions by tandem mass spectrometry led to the identification of many previously characterized mammalian Mediator subunits, as well as a collection of potential
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