Identification of mammalian Mediator subunits with similarities to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3.
The Mediator is a multiprotein coactivator required for activation of RNA polymerase II transcription by DNA binding transactivators. We recently identified a mammalian homologue of yeast Mediator subunit Med8 and 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 purified Med8-containing fractions by tandem mass spectrometry led to the identification of many known mammalian Mediator subunits, as well as four potential Mediator subunits exhibiting sequence similarity to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3. Here we present direct biochemical evidence that these four proteins are bona fide mammalian Mediator subunits. In addition, we identify direct pairwise binding partners of these proteins among the known mammalian Mediator subunits. Taken together, our findings identify a collection of novel mammalian Mediator subunits and shed new light on the underlying architecture of the mammalian Mediator complex.
Sato
Shigeo
S
Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
Tomomori-Sato
Chieri
C
Banks
Charles A S
CA
Sorokina
Irina
I
Parmely
Tari J
TJ
Kong
Stephanie E
SE
Jin
Jingji
J
Cai
Yong
Y
Lane
William S
WS
Brower
Christopher S
CS
Conaway
Ronald C
RC
Conaway
Joan Weliky
JW
D000818:Animals; D008099:Liver / Q000737:chemistry; D013058:Mass Spectrometry; D056892:Mediator Complex; D011233:Precipitin Tests; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012319:RNA Polymerase II; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins; D017385:Sequence Homology; D015534:Trans-Activators / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
C421882:MED8 protein, S cerevisiae; D056892:Mediator Complex; D021122:Protein Subunits; C103856:ROX3 protein, S cerevisiae; C082149:SRB5 protein, S cerevisiae; C082150:SRB6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D012319:RNA Polymerase II