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The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1–13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP–TATA complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation. A sub-nanometre resolution cryo-EM structure of human TFIID bound to TFIIA and core promoter DNA and a model of the TFIID-based pre-initiation complex. Promoter-bound TFIID structure This paper describes the interaction of the human general transcription factor IID (TFIID) with core promoter DNA. TFIID has a central role in the initiation of RNA polymerase II dependent transcription by nucleating assembly of the pre-initiation complex (PIC) at the promoter. Eva Nogales and colleagues determine a sub-nanometre resolution cryo-electron microscopy structure of TFIID bound to TFIIA and core promoter DNA. They also present a cryo-electron microscopy reconstruction of a fully assembled human PIC lacking TBP-associated factors (TAFs). By superimposing common elements between the TFIID–TFIIA–promoter structure and the TAF-less PIC structure, they propose a structural model for the complete TFIID-based PIC, and provide insights into the role of TFIID in promoter recognition, PIC assembly, and transcription initiation.

SAGA (Spt–Ada–Gcn5–acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes 1 . Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å resolution (0.143 Fourier shell correlation). The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP, where steric hindrance represses binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires transcription factor IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding the specific delivery of TBP to gene promoters and the multiple roles of SAGA in regulating gene expression. Structural studies on the yeast transcription coactivator complex SAGA (Spt–Ada–Gcn5–acetyltransferase) provide insights into the mechanism of initiation of regulated transcription by this multiprotein complex, which is conserved among eukaryotes.

Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt–Ada–Gcn5–acetyltransferase) 1 , 2 and transcription factor IID (TFIID) 2 – 4 . SAGA is required for all regulated transcription 5 and is conserved among eukaryotes 6 . SAGA contains four modules 7 – 9 : the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures 10 – 14 , but the structure of the central core module is unknown. Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 Å resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6–Taf9, Taf10–Spt7 and Taf12–Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP) 2 , 7 , 15 – 17 . The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome. Structural studies on the yeast transcription coactivator complex SAGA (Spt–Ada–Gcn5–acetyltransferase) provide insights into the mechanism of initiation of regulated transcription by this multiprotein complex, which is conserved among eukaryotes.

TATA-box binding protein (TBP) is required for every single transcription event in archaea and eukaryotes. It binds DNA and harbors two repeats with an internal structural symmetry that show sequence asymmetry. At various times in evolution, TBP has acquired multiple interaction partners and different organisms have evolved TBP paralogs with additional protein regions. Together, these observations raise questions of what molecular determinants (i.e. key residues) led to the ability of TBP to acquire new interactions, resulting in an increasingly complex transcriptional system in eukaryotes. We present a comprehensive study of the evolutionary history of TBP and its interaction partners across all domains of life, including viruses. Our analysis reveals the molecular determinants and suggests a unified and multi-stage evolutionary model for the functional innovations of TBP. These findings highlight how concerted chemical changes on a conserved structural scaffold allow for the emergence of complexity in a fundamental biological process. The TATA-box binding protein (TBP) is required for transcription initiation in archaea and eukaryotes. Here the authors delineate how TBP’s function has evolved new functional features through context-dependent interactions with various protein partners.

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE ‘extended winged helix’ domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE ‘E-ribbon’ domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein–protein and protein–DNA contacts. The cryo-electron microscopy structures of yeast initiation complexes containing the transcription factors TBP, TFIIA, TFIIB, TFIIE, and TFIIF and containing either closed or open promoter DNA are reported, providing mechanistic insights into DNA opening and template-strand loading. The structural basis of gene initiation The initiation of gene transcription in eukaryotes is tightly controlled at the promoter of each gene through the actions of the pre-initiation complex (PIC), a large multi-subunit composed of general transcription factors, and RNA polymerase II (Pol II) assembles at the promoter to ensure correct loading of Pol II and opening of the duplex DNA for transcription into RNA. Two papers published in this issue report detailed cryo-electron microscopy structures of the Pol II machinery at near-atomic resolution. Eva Nogales and colleagues present structural models of the human PIC at all major steps during transcription initiation at near-atomic resolution. They provide new mechanistic insights into the processes of promoter melting and transcription bubble stabilization, as well as proposing an almost complete structural model of all of the PIC components bound to duplex DNA. Patrick Cramer and colleagues report structures of yeast initiation complexes containing all of the basal transcription factors except TFIIH, and containing either closed or open promoter DNA. They show that DNA opening can occur in the absence of TFIIH, and provide mechanistic insights into DNA opening and template-strand loading. The structures reveal the high structural conservation between yeast and human transcription initiation systems.

Cotton cultivars have evolved to produce extensive, long, seed-born fibers important for the textile industry, but we know little about the molecular mechanism underlying spinnable fiber formation. Here, we report how PACLOBUTRAZOL RESISTANCE 1 (PRE1) in cotton, which encodes a basic helix-loop-helix (bHLH) transcription factor, is a target gene of spinnable fiber evolution. Differential expression of homoeologous genes in polyploids is thought to be important to plant adaptation and novel phenotypes. PRE1 expression is specific to cotton fiber cells, upregulated during their rapid elongation stage and A-homoeologous biased in allotetraploid cultivars. Transgenic studies demonstrated that PRE1 is a positive regulator of fiber elongation. We determined that the natural variation of the canonical TATA-box, a regulatory element commonly found in many eukaryotic core promoters, is necessary for subgenome-biased PRE1 expression, representing a mechanism underlying the selection of homoeologous genes. Thus, variations in the promoter of the cell elongation regulator gene PRE1 have contributed to spinnable fiber formation in cotton. Overexpression of GhPRE1 in transgenic cotton yields longer fibers with improved quality parameters, indicating that this bHLH gene is useful for improving cotton fiber quality.

The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP–exo/seq) 1 , 2 to define this architecture in Saccharomyces cerevisiae . We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI–SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs. A ChIP–exo method is used to define the genome-wide positional organization of proteins associated with gene transcription, DNA replication, centromeres, subtelomeres and transposons, revealing distinct protein assemblies for constitutive and inducible gene expression.

This study focuses on the TATA-box binding, transcription initiation factor TFIID protein, (1tba) and employs a comprehensive approach combining structural, sequence and machine learning-based analysis to investigate its evolutionary relationships. By examining the TATA-box binding like protein, the study aims to identify similarities with other known protein folds, shedding light on its evolutionary relationship, and functional connections. To validate these relationships, a support vector machine (SVM) based algorithm was developed, which was complemented by another machine learning technique random forest, to ensure robust and reliable results. The integrated findings from structural, sequence, and machine learning analysis revealed several domain folds evolutionary related to TATA-box binding protein-like (1tba, B). The SVM-based method developed in this study serves as a valuable tool for identifying novel or functionally similar TATA-box binding proteins, providing deeper insights into their evolutionary and structural relationships. This work not only advances our understanding of the TATA-box binding protein family but also demonstrates the power of integrating computational and machine learning approaches in protein evolution research.

Forces in biological systems are typically investigated at the single-molecule level with atomic force microscopy or optical and magnetic tweezers, but these techniques suffer from limited data throughput and their requirement for a physical connection to the macroscopic world. We introduce a self-assembled nanoscopic force clamp built from DNA that operates autonomously and allows massive parallelization. Single-stranded DNA sections of an origami structure acted as entropic springs and exerted controlled tension in the low piconewton range on a molecular system, whose conformational transitions were monitored by single-molecule Förster resonance energy transfer. We used the conformer switching of a Holliday junction as a benchmark and studied the TATA-binding protein-induced bending of a DNA duplex under tension. The observed suppression of bending above 10 piconewtons provides further evidence of mechanosensitivity in gene regulation.

RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level. Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters. Rengachari et al. provide a structural investigation of Pol II initiation at snRNA gene promoters and find that the snRNA-activating protein complex enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro.