Head Module Control of Mediator Interactions

Takagi, Yuichiro; Calero, Guillermo; Kakemoto, Hirofumi; Brown, Jesse A.; Ehrensberger, Andreas H.; Hudmon, Andy; Asturias, Francisco J.; Kornberg, Roger D.

doi:10.1016/j.molcel.2006.06.007

Cited by 110 publications

(122 citation statements)

References 43 publications

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“…Mediator could then help in overcoming the resulting postinitiation arrest of the PIC potentially by means of its ability to stimulate the CTD kinase activity of TFIIH and concomitant promoter clearance (9,32). This primary effect of DSIF may be amplified in steady-state transcription conditions where committed templates would be prevented from freely reinitiating perhaps owing to the promoter-proximally stalled Pol II.…”

Section: Discussionmentioning

confidence: 99%

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

Malik

Barrero

Jones

2007

Proc. Natl. Acad. Sci. U.S.A.

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The multiprotein Mediator coactivator complex is universally required for transcription of metazoan genes. It has been proposed to function by interfacing between transcriptional activators and the RNA polymerase II machinery. However, in vitro transcription systems reconstituted from homogeneous preparations of RNA polymerase II, the general transcription initiation factors, and the cofactor PC4 display relatively robust activator (HNF-4)-dependent activity, which, nonetheless, can be further stimulated by Mediator. By contrast, an unfractionated nuclear extract-based system in which Mediator has been immunodepleted displays a nearabsolute dependence on ectopic Mediator. Here, we identified and purified an activity, MSA-2, that confers extract-like Mediator responsiveness to our reconstituted system. Mass spectrometric analyses identified its two constituent polypeptides as hSpt5 and hSpt4, which also comprise the elongation factor DSIF. Mechanistically, MSA-2/DSIF acts by restricting overall transcription in the pure system, thereby imposing a strong Mediator dependence. Our data thus point to potential mechanisms for Mediator function beyond its presently believed role in promoting the initial formation of the RNA polymerase II-containing preinitiation complex.DRB sensitivity-inducing factor ͉ hepatocyte nuclear factor-4 ͉ positive cofactor 2 ͉ RNA polymerase II

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Section: Discussionmentioning

confidence: 99%

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

Malik

Barrero

Jones

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…First, the Med18 loop region formed by residues 78-97 interacts with the Med17 CTH of the Fixed jaw domain ( Figure 3a, 3b). Second, the electron density corresponding to the N-terminus of the Med11 subunit (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] indicates an interaction with Med18 (residues 17-27 and 281-289) ( Figure 3c, 3d). The assignment of Med11 residues 1-20 was complicated by the substitution of Met17 to Ser17 (see Methods), and thus an unambiguous sequence marker is lacking.…”

Section: Derivatives (Supplementary Text 3)mentioning

confidence: 99%

“…In vitro transcription assay to assess activity of the recombinant Head module and its mutant form using srb4ts mutant crude extract was described previously 10 . Quantification of transcripts on an absolute scale was performed using a FLA-5100 FUJIFILM fluorescent image analyzer and the MultiGauge software package after addition of 1 nCi of-32 P UTP to the gel 5 min before the end of the run.…”

Section: In Vitro Transcription and The C-terminal Domain (Ctd) Phospmentioning

confidence: 99%

“…In the yeast S. cerevisiae, the Mediator Head module is composed of seven subunits (Med17, Med11, Med22, Med6, Med8, Med18, Med20) 10 . Four subunits are encoded by SRB genes, first identified through a genetic screen for mutations suppressing the Pol II CTD truncation 11,12 .…”

mentioning

confidence: 99%

“…Four subunits are encoded by SRB genes, first identified through a genetic screen for mutations suppressing the Pol II CTD truncation 11,12 . The Head is essential for Mediator function since mutations in the Head module abolish mRNA synthesis in vivo 13,14 and in vitro 15 , and eliminate Mediator interaction with promoters in vivo 10 . The Head module is organized into three domains that can undergo significant conformational changes, and it interacts with the TATA-binding protein (TBP) subunit of general transcription factor TFIID and the Rpb4/Rpb7 Pol II subunits 16 .…”

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confidence: 99%

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Architecture of the mediator head module

Imasaki¹,

Wang²,

Tsai³

et al. 2011

Acta Cryst Sect A

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Mediator is a key regulator of eukaryotic transcription 1 , connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II) 1-4 . In the yeast S. cerevisiae, Mediator comprises 25 subunits with a total mass over 1 MDa 5,6 , and is organized into three modules, termed Head, Middle/Arm and Tail 7-9 . Our understanding of Mediator assembly and its role in regulating transcription has been impeded to date by limited structural information. Here, we report the crystal structure of the essential Mediator Head module (seven subunits, 223 kDa) at 4.3 Å resolution. Our structure reveals three distinct domains with the integrity of the complex centered on a bundle of ten helices from five different Head subunits. An intricate pattern of interactions within this helical bundle ensures stable assembly of the Head subunits, and provides the binding sites for general transcription factors (GTFs) and Pol II. Our structural and functional data suggest the Head module to juxtapose TFIIH and the carboxyl-terminal domain (CTD) of the

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Baculovirus Expression Strategies for Protein Complex Production

Vijayachandran

Viola

Berger

2011

Encyclopedia of Life Sciences

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Protein complexes often contain multiple subunits, and it is these complexes that are at the core of biological function in the cell. Inherently low quantities of protein complexes in eukaryotes can prohibit the extraction of these essential cellular machines from native source material, posing a considerable challenge for functional analysis at the molecular level. The baculovirus expression vector system (BEVS) is particularly useful for recombinantly producing eukaryotic proteins in the quality and quantity required for deciphering their structure and function. Recent efforts have focused on improving reagents and streamlining protocols to routinely express protein complexes by recombinant baculoviruses. The challenge of integration of baculovirus expression in high‐throughput production pipelines for proteins and their complexes is being addressed. Key Concepts: Protein complexes are a cornerstone of biological function in the cell. Recombinant production is essential to study many multiprotein complexes. Ongoing development of expression systems for producing multiprotein complexes. BEVS as a method of choice for large‐scale production of complexes requires integration of all genes in a single multigene baculovirus rather than coinfecting insect cell cultures with many baculoviruses. Creation of new reagents and streamlined protocols to address the challenge of generating multigene baculoviruses for routine laboratory use. Engineering baculoviruses and new host cell lines to further enhance protein production by BEVS. Multigene vector generation incorporated into automation pipelines to increase the throughput of protein complex expression by BEVS.

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Head Module Control of Mediator Interactions

Cited by 110 publications

References 43 publications

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

Architecture of the mediator head module

Baculovirus Expression Strategies for Protein Complex Production

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