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Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the tricarboxylic acid (TCA) cycle. Here we show that the deglycase DJ-1 (encoded by PARK7, a key familial Parkinson's disease gene) is a pacemaker regulating PDH activity in CD4 + regulatory T cells (T reg cells). DJ-1 binds to PDHE1-β (PDHB), inhibiting phosphorylation of PDHE1-α (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Park7 (Dj-1) deletion impairs T reg survival starting in young mice and reduces T reg homeostatic proliferation and cellularity only in aged mice. This leads to increased severity in aged mice during the remission of experimental autoimmune encephalomyelitis (EAE). Dj-1 deletion also compromises differentiation of inducible T reg cells especially in aged mice, and the impairment occurs via regulation of PDHB. These findings provide unforeseen insight into the complicated regulatory machinery of the PDH complex. As T reg homeostasis is dysregulated in many complex diseases, the DJ-1-PDHB axis represents a potential target to maintain or re-establish T reg homeostasis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled in vitro constituting a building block for the further assembly of either lobe A or B in TFIID. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP., (Copyright © 2020 American Society for Microbiology.)

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility. The Saccharomyces cerevisiae RSC ( R emodeling the S tructure of C hromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored to this core by the N-terminus of Sth1. Our cryo-EM analysis of RSC bound to a nucleosome core particle shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of the Rsc8 SWIRM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes., Competing Interests: AP, CM, BG, JL, SZ, JR, EN No competing interests declared, (© 2019, Patel et al.)

Chemical cross-linking combined with mass spectrometry (CL-MS) is a powerful method for characterizing the architecture of protein assemblies and for mapping protein-protein interactions. Despite its proven utility, confident identification of cross-linked peptides remains a formidable challenge, especially when the peptides are derived from complex mixtures. MS cleavable cross-linkers are gaining importance for CL-MS as they permit reliable identification of cross-linked peptides by whole proteome database searching using MS/MS information. Here we introduce a novel class of MS cleavable cross-linkers called isotopomeric cross-linkers (ICLs), which allow for confident and efficient identification of cross-linked peptides by whole proteome database searching. ICLs are simple, symmetrical molecules that asymmetrically incorporate heavy and light stable isotopes into the two arms of the cross-linker. As a result of this property, ICLs automatically generate pairs of isotopomeric cross-linked peptides, which differ only by the positions of the heavy and light isotopes. Upon fragmentation during MS analysis, these isotopomeric cross-linked peptides generate unique isotopic doublet ions that correspond to the individual peptides in the cross-link. The doublet ion information is used to determine the masses of the two cross-linked peptides from the same MS2 spectrum that is also used for peptide spectrum matching (PSM) by sequence database searching. Here we present the rationale for and mechanism of cross-linked peptide identification by ICL-MS. We describe the synthesis of the ICL-1 reagent, the ICL-MS workflow, and the performance characteristics of ICL-MS for identifying cross-linked peptides derived from increasingly complex mixtures by whole proteome database searching.

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The general transcription factor IID (TFIID) is a critical component of the eukaryotic transcription preinitiation complex (PIC) and is responsible for recognizing the core promoter DNA and initiating PIC assembly. We used cryo-electron microscopy, chemical cross-linking mass spectrometry, and biochemical reconstitution to determine the complete molecular architecture of TFIID and define the conformational landscape of TFIID in the process of TATA box-binding protein (TBP) loading onto promoter DNA. Our structural analysis revealed five structural states of TFIID in the presence of TFIIA and promoter DNA, showing that the initial binding of TFIID to the downstream promoter positions the upstream DNA and facilitates scanning of TBP for a TATA box and the subsequent engagement of the promoter. Our findings provide a mechanistic model for the specific loading of TBP by TFIID onto the promoter., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. Here we present the cryo-electron microscopy structure of promoter-bound yeast TFIID at a resolution better than 5 Å, except for a flexible domain. We position the crystal structures of several subunits and, in combination with cross-linking studies, describe the quaternary organization of TFIID. The compact tri lobed architecture is stabilized by a topologically closed Taf5-Taf6 tetramer. We confirm the unique subunit stoichiometry prevailing in TFIID and uncover a hexameric arrangement of Tafs containing a histone fold domain in the Twin lobe.

Eukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While the AD function of both factors depended on hydrophobic residues, Ino2 further required key conserved acidic and polar residues for optimal function. Binding studies showed that the ADs bound multiple Med15 activator-binding domains (ABDs) with similar orders of micromolar affinity and similar but distinct thermodynamic properties. Protein cross-linking data show that no unique complex was formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators., (Copyright © 2018 American Society for Microbiology.)

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

TFIIH is a 10-subunit RNA polymerase II basal transcription factor with a dual role in DNA repair. TFIIH contains three enzymatic functions and over 30 conserved subdomains and topological regions. We systematically tested the function of these regions in three TFIIH core module subunits, i.e., Ssl1, Tfb4, and Tfb2, in the DNA translocase subunit Ssl2, and in the kinase module subunit Tfb3. Our results are consistent with previously predicted roles for the Tfb2 Hub, Ssl2 Lock, and Tfb3 Latch regions, with mutations in these elements typically having severe defects in TFIIH subunit association. We also found unexpected roles for other domains whose function had not previously been defined. First, the Ssl1-Tfb4 Ring domains are important for TFIIH assembly. Second, the Tfb2 Hub and HEAT domains have an unexpected role in association with Tfb3. Third, the Tfb3 Ring domain is important for association with many other TFIIH subunits. Fourth, a partial deletion of the Ssl1 N-terminal extension (NTE) domain inhibits TFIIH function without affecting subunit association. Finally, we used site-specific cross-linking to localize the Tfb3-binding surface on the Rad3 Arch domain. Our cross-linking results suggest that Tfb3 and Rad3 have an unusual interface, with Tfb3 binding on two opposite faces of the Arch., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit., (Copyright © 2015 Elsevier Inc. All rights reserved.)

ATP-dependent chromatin remodeling complexes alter chromatin structure through interactions with chromatin substrates such as DNA, histones, and nucleosomes. However, whether chromatin remodeling complexes have the ability to regulate nonchromatin substrates remains unclear. Saccharomyces cerevisiae checkpoint kinase Mec1 (ATR in mammals) is an essential master regulator of genomic integrity. Here we found that the SWI/SNF chromatin remodeling complex is capable of regulating Mec1 kinase activity. In vivo, Mec1 activity is reduced by the deletion of Snf2, the core ATPase subunit of the SWI/SNF complex. SWI/SNF interacts with Mec1, and cross-linking studies revealed that the Snf2 ATPase is the main interaction partner for Mec1. In vitro, SWI/SNF can activate Mec1 kinase activity in the absence of chromatin or known activators such as Dpb11. The subunit requirement of SWI/SNF-mediated Mec1 regulation differs from that of SWI/SNF-mediated chromatin remodeling. Functionally, SWI/SNF-mediated Mec1 regulation specifically occurs in S phase of the cell cycle. Together, these findings identify a novel regulator of Mec1 kinase activity and suggest that ATP-dependent chromatin remodeling complexes can regulate nonchromatin substrates such as a checkpoint kinase., (© 2015 Kapoor et al.; Published by Cold Spring Harbor Laboratory Press.)

The conserved transcription coactivator SAGA is comprised of several modules that are involved in activator binding, TBP binding, histone acetylation (HAT) and deubiquitination (DUB). Crosslinking and mass spectrometry, together with genetic and biochemical analyses, were used to determine the molecular architecture of the SAGA-TBP complex. We find that the SAGA Taf and Taf-like subunits form a TFIID-like core complex at the center of SAGA that makes extensive interactions with all other SAGA modules. SAGA-TBP binding involves a network of interactions between subunits Spt3, Spt8, Spt20, and Spt7. The HAT and DUB modules are in close proximity, and the DUB module modestly stimulates HAT function. The large activator-binding subunit Tra1 primarily connects to the TFIID-like core via its FAT domain. These combined results were used to derive a model for the arrangement of the SAGA subunits and its interactions with TBP. Our results provide new insight into SAGA function in gene regulation, its structural similarity with TFIID, and functional interactions between the SAGA modules., (© 2014 The Authors.)

Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5' GCTGGTGG 3') during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3'-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular "signal transduction" from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Core Factor (CF) is a conserved RNA polymerase (Pol) I general transcription factor comprising Rrn6, Rrn11 and the TFIIB-related subunit Rrn7. CF binds TATA-binding protein (TBP), Pol I and the regulatory factors Rrn3 and upstream activation factor. We used chemical cross-linking-MS to determine the molecular architecture of CF and its interactions with TBP. The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Pol I factor SL1. We validated the cross-linking-derived model through a series of genetic and biochemical assays. Our combined results show the architecture of CF and the functions of the CF subunits in assembly of the complex. We extend these findings to model how CF assembles into the Pol I preinitiation complex, providing new insight into the roles of CF, TBP and Rrn3.

Subunits of mammalian SWI/SNF (mSWI/SNF or BAF) complexes have recently been implicated as tumor suppressors in human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of endogenous mSWI/SNF complexes, which identified several new dedicated, stable subunits not found in yeast SWI/SNF complexes, including BCL7A, BCL7B and BCL7C, BCL11A and BCL11B, BRD9 and SS18. Incorporating these new members, we determined mSWI/SNF subunit mutation frequency in exome and whole-genome sequencing studies of primary human tumors. Notably, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, mutations affecting more than one subunit, defined here as compound heterozygosity, are prevalent in certain cancers. Our studies demonstrate that mSWI/SNF is the most frequently mutated chromatin-regulatory complex (CRC) in human cancer, exhibiting a broad mutation pattern, similar to that of TP53. Thus, proper functioning of polymorphic BAF complexes may constitute a major mechanism of tumor suppression.

Regulation of gene expression involves the orchestrated interaction of a large number of proteins with transcriptional regulatory elements in the context of chromatin. Our understanding of gene regulation is limited by the lack of a protein measurement technology that can systematically detect and quantify the ensemble of proteins associated with the transcriptional regulatory elements of specific genes. Here, we introduce a set of selected reaction monitoring (SRM) assays for the systematic measurement of 464 proteins with known or suspected roles in transcriptional regulation at RNA polymerase II transcribed promoters in Saccharomyces cerevisiae. Measurement of these proteins in nuclear extracts by SRM permitted the reproducible quantification of 42% of the proteins over a wide range of abundances. By deploying the assay to systematically identify DNA binding transcriptional regulators that interact with the environmentally regulated FLO11 promoter in cell extracts, we identified 15 regulators that bound specifically to distinct regions along ∼600 bp of the regulatory sequence. Importantly, the dataset includes a number of regulators that have been shown to either control FLO11 expression or localize to these regulatory regions in vivo. We further validated the utility of the approach by demonstrating that two of the SRM-identified factors, Mot3 and Azf1, are required for proper FLO11 expression. These results demonstrate the utility of SRM-based targeted proteomics to guide the identification of gene-specific transcriptional regulators.

The largest transcription factor IID (TFIID) subunit, TBP-associated factor 1 (TAF1), possesses protein kinase and histone acetyltransferase (HAT) activities. Both enzymatic activities are essential for transcription from a subset of genes and G(1) progression in mammalian cells. TAF7, another TFIID subunit, binds TAF1 and inhibits TAF1 HAT activity. Here we present data demonstrating that disruption of the TAF1/TAF7 interaction within TFIID by protein phosphorylation leads to activation of TAF1 HAT activity and stimulation of cyclin D1 and cyclin A gene transcription. Overexpression and small interfering RNA knockdown experiments confirmed that TAF7 functions as a transcriptional repressor at these promoters. Release of TAF7 from TFIID by TAF1 phosphorylation of TAF7 increased TAF1 HAT activity and elevated histone H3 acetylation levels at the cyclin D1 and cyclin A promoters. Serine-264 of TAF7 was identified as a substrate for TAF1 kinase activity. Using TAF7 S264A and S264D phosphomutants, we determined that the phosphorylation state of TAF7 at S264 influences the levels of cyclin D1 and cyclin A gene transcription and promoter histone H3 acetylation. Our studies have uncovered a novel function for the TFIID subunit TAF7 as a phosphorylation-dependent regulator of TAF1-catalyzed histone H3 acetylation at the cyclin D1 and cyclin A promoters.

Knowledge of protein structures and protein-protein interactions is essential for understanding biological processes. Chemical cross-linking combined with mass spectrometry is an attractive approach for studying protein-protein interactions and protein structure, but to date its use has been limited largely by low yields of informative cross-links (because of inefficient cross-linking reactions) and by the difficulty of confidently identifying the sequences of cross-linked peptide pairs from their fragmentation spectra. Here we present an approach based on a new MS labile cross-linking reagent, BDRG (biotin-aspartate-Rink-glycine), which addresses these issues. BDRG incorporates a biotin handle (for enrichment of cross-linked peptides prior to MS analysis), two pentafluorophenyl ester groups that react with peptide amines, and a labile Rink-based bond between the pentafluorophenyl groups that allows cross-linked peptides to be separated during MS and confidently identified by database searching of their fragmentation spectra. We developed a protocol for the identification of BDRG cross-linked peptides derived from purified or partially purified protein complexes, including software to aid in the identification of different classes of cross-linker-modified peptides. Importantly, our approach permits the use of high accuracy precursor mass measurements to verify the database search results. We demonstrate the utility of the approach by applying it to purified yeast TFIIE, a heterodimeric transcription factor complex, and to a single-step affinity-purified preparation of the 12-subunit RNA polymerase II complex. The results show that the method is effective at identifying cross-linked peptides derived from purified and partially purified protein complexes and provides complementary information to that from other structural approaches. As such, it is an attractive approach to study the topology of protein complexes.

Biomedical research requires protein detection technology that is not only sensitive and quantitative, but that can reproducibly measure any set of proteins in a biological system in a high throughput manner. Here we report the development and application of a targeted proteomics platform termed index-ion triggered MS2 ion quantification (iMSTIQ) that allows reproducible and accurate peptide quantification in complex mixtures. The key feature of iMSTIQ is an approach called index-ion triggered analysis (ITA) that permits the reproducible acquisition of full MS2 spectra of targeted peptides independent of their ion intensities. Accurate quantification is achieved by comparing the relative intensities of multiple pairs of fragment ions derived from isobaric targeted peptides during MS2 analysis. Importantly, the method takes advantage of the favorable performance characteristics of the LTQ-Orbitrap, which include high mass accuracy, resolution, and throughput. As such it provides an attractive targeted proteomics tool to meet the demands of systems biology research and biomarker studies.

The Saccharomyces cerevisiae kinase Bur1 is involved in coupling transcription elongation to chromatin modification, but not all important Bur1 targets in the elongation complex are known. Using a chemical genetics strategy wherein Bur1 kinase was engineered to be regulated by a specific inhibitor, we found that Bur1 phosphorylates the Spt5 C-terminal repeat domain (CTD) both in vivo and in isolated elongation complexes in vitro. Deletion of the Spt5 CTD or mutation of the Spt5 serines targeted by Bur1 reduces recruitment of the PAF complex, which functions to recruit factors involved in chromatin modification and mRNA maturation to elongating polymerase II (Pol II). Deletion of the Spt5 CTD showed the same defect in PAF recruitment as rapid inhibition of Bur1 kinase activity, and this Spt5 mutation led to a decrease in histone H3K4 trimethylation. Brief inhibition of Bur1 kinase activity in vivo also led to a significant decrease in phosphorylation of the Pol II CTD at Ser-2, showing that Bur1 also contributes to Pol II Ser-2 phosphorylation. Genetic results suggest that Bur1 is essential for growth because it targets multiple factors that play distinct roles in transcription.

Mammalian SWI/SNF [also called BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodeling complexes are essential for formation of the totipotent and pluripotent cells of the early embryo. In addition, subunits of this complex have been recovered in screens for genes required for nuclear reprogramming in Xenopus and mouse embryonic stem cell (ES) morphology. However, the mechanism underlying the roles of these complexes is unclear. Here, we show that BAF complexes are required for the self-renewal and pluripotency of mouse ES cells but not for the proliferation of fibroblasts or other cells. Proteomic studies reveal that ES cells express distinctive complexes (esBAF) defined by the presence of Brg (Brahma-related gene), BAF155, and BAF60A, and the absence of Brm (Brahma), BAF170, and BAF60C. We show that this specialized subunit composition is required for ES cell maintenance and pluripotency. Our proteomic analysis also reveals that esBAF complexes interact directly with key regulators of pluripotency, suggesting that esBAF complexes are specialized to interact with ES cell-specific regulators, providing a potential explanation for the requirement of BAF complexes in pluripotency.

Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Although such analyses typically assume that a protein's peptide fragments are observed with equal likelihood, only a few so-called 'proteotypic' peptides are repeatedly and consistently identified for any given protein present in a mixture. Using >600,000 peptide identifications generated by four proteomic platforms, we empirically identified >16,000 proteotypic peptides for 4,030 distinct yeast proteins. Characteristic physicochemical properties of these peptides were used to develop a computational tool that can predict proteotypic peptides for any protein from any organism, for a given platform, with >85% cumulative accuracy. Possible applications of proteotypic peptides include validation of protein identifications, absolute quantification of proteins, annotation of coding sequences in genomes, and characterization of the physical principles governing key elements of mass spectrometric workflows (e.g., digestion, chromatography, ionization and fragmentation).

We demonstrate an integrated approach to build, test, and refine a model of a cellular pathway, in which perturbations to critical pathway components are analyzed using DNA microarrays, quantitative proteomics, and databases of known physical interactions. Using this approach, we identify 997 messenger RNAs responding to 20 systematic perturbations of the yeast galactose-utilization pathway, provide evidence that approximately 15 of 289 detected proteins are regulated posttranscriptionally, and identify explicit physical interactions governing the cellular response to each perturbation. We refine the model through further iterations of perturbation and global measurements, suggesting hypotheses about the regulation of galactose utilization and physical interactions between this and a variety of other metabolic pathways.

High levels of gene transcription by RNA polymerase II depend on high rates of transcription initiation and reinitiation. Initiation requires recruitment of the complete transcription machinery to a promoter, a process facilitated by activators and chromatin remodelling factors. Reinitiation probably occurs through a different pathway. After initiation, a subset of the transcription machinery remains at the promoter, forming a platform for assembly of a second transcription complex. Here we describe the isolation of a reinitiation intermediate that includes transcription factors TFIID, TFIIA, TFIIH, TFIIE and Mediator. This intermediate can act as a scaffold for formation of a functional reinitiation complex. Formation of this scaffold is dependent on ATP and TFIIH. The scaffold is stabilized in the presence of the activator Gal4-VP16, but not Gal4-AH, suggesting a new role for some activators and Mediator in promoting high levels of transcription.

The yeast transcription factor IIA (TFIIA), a component of the basal transcription machinery of RNA polymerase II and implicated in vitro in regulation of basal transcription, is composed of two subunits of 32 and 13.5 kilodaltons. The genes that encode these subunits, termed TOA1 and TOA2, respectively, were cloned. Neither gene shares obvious sequence similarity with the other or with any other previously identified genes. The recombinant factor bound to a TATA binding protein-DNA complex and complemented yeast and mammalian in vitro transcription systems depleted of TFIIA. Both the TOA1 and TOA2 genes are essential for growth of yeast.

The general transcription factor TFIIA was purified from yeast. A key step in the purification was affinity chromatography using a column containing the adenovirus major late promoter with bound recombinant TFIID to which TFIIA binds with high affinity. TFIIA activity copurifies with two polypeptides of molecular mass 32 and 13.5 kDa. Elution and renaturation of these two polypeptides from sodium dodecyl sulfate-polyacrylamide gels showed that both polypeptides were required for TFIIA activity. TFIIA activity was measured by both a native gel shift assay and by in vitro complementation of transcription using yeast nuclear extracts depleted of TFIIA. The purified renatured yeast TFIIA also complements basal level transcription using a mammalian transcription system depleted of TFIIA. Native TFIIA has an apparent molecular mass of approximately 90 kDa measured by gel filtration chromatography. TFIIA binds to a TFIID.TATA element.DNA complex with an apparent equilibrium dissociation constant (KD) of 20 pM. This affinity is about 100-fold greater than the affinity of TFIID for TATA elements and much greater than the affinity of TFIIA for TFIID not bound to DNA.