Role for the MED21-MED7 Hinge in Assembly of the Mediator-RNA Polymerase II Holoenzyme.
Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved "hinge" in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly.
From the Stowers Institute for Medical Research, Kansas City, Missouri 64110.
Shigeo
S
Sato
From the Stowers Institute for Medical Research, Kansas City, Missouri 64110.
Chieri
C
Tomomori-Sato
the Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, California 92037, and.
Kuang-Lei
KL
Tsai
the Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, California 92037, and.
Xiaodi
X
Yu
From the Stowers Institute for Medical Research, Kansas City, Missouri 64110.
Mihaela
M
Sardiu
From the Stowers Institute for Medical Research, Kansas City, Missouri 64110.
Anita
A
Saraf
From the Stowers Institute for Medical Research, Kansas City, Missouri 64110.|the Departments of Pathology and Laboratory Medicine and.
Michael P
MP
Washburn
From the Stowers Institute for Medical Research, Kansas City, Missouri 64110.
Laurence
L
Florens
the Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, California 92037, and.
Francisco J
FJ
Asturias
From the Stowers Institute for Medical Research, Kansas City, Missouri 64110.|Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160.
Ronald C
RC
Conaway
From the Stowers Institute for Medical Research, Kansas City, Missouri 64110, jlc@stowers.org.|Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160.
Joan W
http://orcid.org/0000-0002-2786-0663
JW
Conaway
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D056892:Mediator Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D020035:Holoenzymes; C098617:MED21 protein, human; C492153:MED7 protein, human; D056892:Mediator Complex; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II
Mediator; RNA polymerase II; general transcription factor (GTF); transcription; transcription coregulator; transcription regulation