Martin Seizl

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

To initiate gene transcription, RNA polymerase II (Pol II) requires the transcription factor IIB (B). Here we present the crystal structure of the complete Pol II–B complex at 4.3 Å resolution, and complementary functional data. The results indicate the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active centre cleft with the ‘B-core’ domain that binds the wall at the end of the cleft. DNA is then opened with the help of the ‘B-linker’ that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the ‘B-reader’ that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger B release and elongation complex formation.

Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are fivefold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4-Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.

Structure of the Mediator head module

Gene transcription by RNA polymerase (Pol) II requires the coactivator complex Mediator. Mediator connects transcriptional regulators and Pol II, and is linked to human disease. Mediator from the yeast Saccharomyces cerevisiae has a molecular mass of 1.4 megadaltons and comprises 25 subunits that form the head, middle, tail and kinase modules. The head module constitutes one-half of the essential Mediator core, and comprises the conserved subunits Med6, Med8, Med11, Med17, Med18, Med20 and Med22. Recent X-ray analysis of the S. cerevisiae head module at 4.3 Å resolution led to a partial architectural model with three submodules called neck, fixed jaw and moveable jaw. Here we determine de novo the crystal structure of the head module from the fission yeast Schizosaccharomyces pombe at 3.4 Å resolution. Structure solution was enabled by new structures of Med6 and the fixed jaw, and previous structures of the moveable jaw and part of the neck, and required deletion of Med20. The S. pombe head module resembles the head of a crocodile with eight distinct elements, of which at least four are mobile. The fixed jaw comprises tooth and nose domains, whereas the neck submodule contains a helical spine and one limb, with shoulder, arm and finger elements. The arm and the essential shoulder contact other parts of Mediator. The jaws and a central joint are implicated in interactions with Pol II and its carboxy-terminal domain, and the joint is required for transcription in vitro. The S. pombe head module structure leads to a revised model of the S. cerevisiae module, reveals a high conservation and flexibility, explains known mutations, and provides the basis for unravelling a central mechanism of gene regulation.

Structure and VP16 binding of the Mediator Med25 activator interaction domain

Eukaryotic transcription is regulated by interactions between gene-specific activators and the coactivator complex Mediator. Here we report the NMR structure of the Mediator subunit Med25 (also called Arc92) activator interaction domain (ACID) and analyze the structural and functional interaction of ACID with the archetypical acidic transcription activator VP16. Unlike other known activator targets, ACID forms a seven-stranded β-barrel framed by three helices. The VP16 subdomains H1 and H2 bind to opposite faces of ACID and cooperate during promoter-dependent activated transcription in a in vitro system. The activator-binding ACID faces are functionally required and conserved among higher eukaryotes. Comparison with published activator structures reveals that the VP16 activation domain uses distinct interaction modes to adapt to unrelated target surfaces and folds that evolved for activator binding.

A structural perspective on Mediator function

Gene transcription by RNA polymerase II requires the multiprotein coactivator complex Mediator. Mediator was identified two decades ago, but its molecular mechanisms remain poorly understood, because structural studies are hampered by its large size, modularity, and flexibility. Here we collect all available structural data on Mediator and discuss their functional implications. Progress was made in understanding the interactions of Mediator with gene-specific transcriptional regulators and the general transcription machinery. However, around 80% of the Mediator structure remains unknown and details on the Mediator-Pol II interface are lacking. In the future, an integrated structural biology approach may unravel the functional architecture of Mediator-regulated promoter assemblies and holds the promise of understanding a key mechanism of gene regulation.

Iwr1 Directs RNA Polymerase II Nuclear Import

RNA polymerase (Pol) II transcribes protein-coding genes in the nucleus of eukaryotic cells and consists of 12 polypeptide subunits. It is unknown how Pol II is imported into the nucleus. Here we show that Pol II nuclear import requires the protein Iwr1 and provide evidence for cyclic Iwr1 function. Iwr1 binds Pol II in the active center cleft between the two largest subunits, maybe facilitating or sensing complete Pol II assembly in the cytoplasm. Iwr1 then uses an N-terminal bipartite nuclear localization signal that is recognized by karyopherin α to direct Pol II nuclear import. In the nucleus, Iwr1 is displaced from Pol II by transcription initiation factors and nucleic acids, enabling its export and recycling. Iwr1 function is Pol II specific, transcription independent, and apparently conserved from yeast to human.

Identification, structure, and functional requirement of the Mediator submodule Med7N/31

Mediator is a modular multiprotein complex required for regulated transcription by RNA polymerase (Pol) II. Here, we show that the middle module of the Mediator core contains a submodule of unique structure and function that comprises the N‐terminal part of subunit Med7 (Med7N) and the highly conserved subunit Med31 (Soh1). The Med7N/31 submodule shows a conserved novel fold, with two proline‐rich stretches in Med7N wrapping around the right‐handed four‐helix bundle of Med31. In vitro, Med7N/31 is required for activated transcription and can act in trans when added exogenously. In vivo, Med7N/31 has a predominantly positive function on the expression of a specific subset of genes, including genes involved in methionine metabolism and iron transport. Comparative phenotyping and transcriptome profiling identify specific and overlapping functions of different Mediator submodules.

Mediator head subcomplex Med11/22 contains a common helix bundle building block with a specific function in transcription initiation complex stabilization

Mediator is a multiprotein co-activator of RNA polymerase (Pol) II transcription. Mediator contains a conserved core that comprises the ‘head’ and ‘middle’ modules. We present here a structure–function analysis of the essential Med11/22 heterodimer, a part of the head module. Med11/22 forms a conserved four-helix bundle domain with C-terminal extensions, which bind the central head subunit Med17. A highly conserved patch on the bundle surface is required for stable transcription pre-initiation complex formation on a Pol II promoter in vitro and in vivo and may recruit the general transcription factor TFIIH. The bundle domain fold is also present in the Mediator middle module subcomplex Med7/21 and is predicted in the Mediator heterodimers Med2/3, Med4/9, Med10/14 and Med28/30. The bundle domain thus represents a common building block that has been multiplied and functionally diversified during Mediator evolution in eukaryotes.

Structure–system correlation identifies a gene regulatory Mediator submodule

A combination of crystallography, biochemistry, and gene expression analysis identifies the coactivator subcomplex Med8C/18/20 as a functionally distinct submodule of the Mediator head module. Med8C forms a conserved alpha-helix that tethers Med18/20 to the Mediator. Deletion of Med8C in vivo results in dissociation of Med18/20 from Mediator and in loss of transcription activity of extracts. Deletion of med8C, med18, or med20 causes similar changes in the yeast transcriptome, establishing Med8C/18/20 as a predominantly positive, gene-specific submodule required for low transcription levels of nonactivated genes, including conjugation genes. The presented structure-based system perturbation is superior to gene deletion analysis of gene regulation.

Model of the Mediator middle module based on protein cross-linking

The essential core of the transcription coactivator Mediator consists of two conserved multiprotein modules, the head and middle modules. Whereas the structure of the head module is known, the structure of the middle module is lacking. Here we report a 3D model of a 6-subunit Mediator middle module. The model was obtained by arranging crystal structures and homology models of parts of the module based on lysine–lysine cross-links obtained by mass spectrometric analysis. The model contains a central tetramer formed by the heterodimers Med4/Med9 and Med7/Med21. The Med7/Med21 heterodimer is flanked by subunits Med10 and Med31. The model is highly extended, suggests that the middle module is flexible and contributes to a molecular basis for detailed structure–function studies of RNA polymerase II regulation.