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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.

RNA polymerase (RNAP) frequently pauses during the transcription of DNA to RNA to regulate gene expression. Transcription factors NusA and NusG modulate pausing, have opposing roles, but can bind RNAP simultaneously. Here we report cryo-EM reconstructions of Escherichia coli RNAP bound to NusG, or NusA, or both. RNAP conformational changes, referred to as swivelling, correlate with transcriptional pausing. NusA facilitates RNAP swivelling to further increase pausing, while NusG counteracts this role. Their structural effects are consistent with biochemical results on two categories of transcriptional pauses. In addition, the structures suggest a cooperative mechanism of NusA and NusG during Rho-mediated transcription termination. Our results provide a structural rationale for the stochastic nature of pausing and termination and how NusA and NusG can modulate it. Pausing of RNA polymerase (RNAP) and transcription is regulated by the NusA and NusG transcription factors in bacteria. Here the authors provide structural evidence for how they interact with RNAP to carry out their pausing roles and also reveal functions for NusA and NusG in transcription termination.

Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation. Eukaryotic RNA polymerase I (Pol I) is responsible for the transcription of rRNA genes. Here the authors determine the cryo-EM structure of the Pol I-Rrn3 complex, providing insight into how Rrn3 stabilizes the monomeric initiation competent Pol I to drive pre-initiation complex formation.

The ribosome maturation factor Rea1 (or Midasin) catalyses the removal of assembly factors from large ribosomal subunit precursors and promotes their export from the nucleus to the cytosol. Rea1 consists of nearly 5000 amino-acid residues and belongs to the AAA+ protein family. It consists of a ring of six AAA+ domains from which the ≈1700 amino-acid residue linker emerges that is subdivided into stem, middle and top domains. A flexible and unstructured D/E rich region connects the linker top to a MIDAS (metal ion dependent adhesion site) domain, which is able to bind the assembly factor substrates. Despite its key importance for ribosome maturation, the mechanism driving assembly factor removal by Rea1 is still poorly understood. Here we demonstrate that the Rea1 linker is essential for assembly factor removal. It rotates and swings towards the AAA+ ring following a complex remodelling scheme involving nucleotide independent as well as nucleotide dependent steps. ATP-hydrolysis is required to engage the linker with the AAA+ ring and ultimately with the AAA+ ring docked MIDAS domain. The interaction between the linker top and the MIDAS domain allows direct force transmission for assembly factor removal. The ribosome maturation factor Rea1 catalyzes essential steps in the production of the large ribosomal subunit. Here, Busselez et al. describe functionally important conformational changes in Rea1.

The mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2 RW/+ and Dnm2 RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2 RW/+ mice and rescued the perinatal lethality and survival of Dnm2 RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations.

Dynamin 2 mechanoenzyme is a key regulator of membrane remodeling and gain-of-function mutations in its gene cause centronuclear myopathies. Here, we investigate the functions of dynamin 2 isoforms and their associated phenotypes and, specifically, the ubiquitous and muscle-specific dynamin 2 isoforms expressed in skeletal muscle. In cell-based assays, we show that a centronuclear myopathy-related mutation in the ubiquitous but not the muscle-specific dynamin 2 isoform causes increased membrane fission. In vivo, overexpressing the ubiquitous dynamin 2 isoform correlates with severe forms of centronuclear myopathy, while overexpressing the muscle-specific isoform leads to hallmarks seen in milder cases of the disease. Previous mouse studies suggested that reduction of the total dynamin 2 pool could be therapeutic for centronuclear myopathies. Here, dynamin 2 splice switching from muscle-specific to ubiquitous dynamin 2 aggravated the phenotype of a severe X-linked form of centronuclear myopathy caused by loss-of-function of the MTM1 phosphatase, supporting the importance of targeting the ubiquitous isoform for efficient therapy in muscle. Our results highlight that the ubiquitous and not the muscle-specific dynamin 2 isoform is the main modifier contributing to centronuclear myopathy pathology. Dynamin 2 is a large GTPase linked to several human diseases. Here, Gómez-Oca et al. investigate the functions of muscle dynamin 2 isoforms and provide insights into their differential implication in centronuclear myopathy pathogenesis and treatment.

Integration of the human immunodeficiency virus (HIV‐1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild‐type full‐length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA‐binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral‐induced pathologies.

Integration of the HIV-1 cDNA into the human genome is catalyzed by the viral integrase (IN) protein. Several studies have shown the importance of cellular cofactors that interact with integrase and affect viral integration and infectivity. In this study, we produced a stable complex between HIV-1 integrase, viral U5 DNA, the cellular cofactor LEDGF/p75 and the integrase binding domain of INI1 (INI1-IBD), a subunit of the SWI/SNF chromatin remodeling factor. The stoichiometry of the IN/LEDGF/INI1-IBD/DNA complex components was found to be 4/2/2/2 by mass spectrometry and Fluorescence Correlation Spectroscopy. Functional assays showed that INI1-IBD inhibits the 3' processing reaction but does not interfere with specific viral DNA binding. Integration assays demonstrate that INI1-IBD decreases the amount of integration events but inhibits by-product formation such as donor/donor or linear full site integration molecules. Cryo-electron microscopy locates INI1-IBD within the cellular DNA binding site of the IN/LEDGF complex, constraining the highly flexible integrase in a stable conformation. Taken together, our results suggest that INI1 could stabilize the PIC in the host cell, by maintaining integrase in a stable constrained conformation which prevents non-specific interactions and auto integration on the route to its integration site within nucleosomes, while LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vDNA integration. Moreover, our results provide the basis for a novel type of integrase inhibitor (conformational inhibitor) representing a potential new strategy for use in human therapy.

Chromatin structure is a key regulator of DNA transcription, replication and repair 1 . In humans, the TIP60–EP400 complex (TIP60-C) is a 20-subunit assembly that affects chromatin structure through two enzymatic activities: ATP-dependent exchange of histone H2A–H2B for H2A.Z–H2B, and histone acetylation. In yeast, however, these activities are performed by two independent complexes—SWR1 and NuA4, respectively 2 , 3 . How the activities of the two complexes are merged into one supercomplex in humans, and what this association entails for the structure and mechanism of the proteins and their recruitment to chromatin, are unknown. Here we describe the structure of the endogenous human TIP60-C. We find a three-lobed architecture composed of SWR1-like (SWR1L) and NuA4-like (NuA4L) parts, which associate with a TRRAP activator-binding module. The huge EP400 subunit contains the ATPase motor, traverses the junction between SWR1L and NuA4L twice and constitutes the scaffold of the three-lobed architecture. NuA4L is completely rearranged compared with its yeast counterpart. TRRAP is flexibly tethered to NuA4L—in stark contrast to its robust connection to the completely opposite side of NuA4 in yeast 4 – 7 . A modelled nucleosome bound to SWR1L, supported by tests of TIP60-C activity, suggests that some aspects of the histone exchange mechanism diverge from what is seen in yeast 8 , 9 . Furthermore, a fixed actin module (as opposed to the mobile actin subcomplex in SWR1; ref. 8 ), the flexibility of TRRAP and the weak effect of extranucleosomal DNA on exchange activity lead to a different, activator-based mode of enlisting TIP60-C to chromatin. The structure of human TIP60-C uncovers a molecular machine that modifies and exchanges histones in the nucleosome, illustrating how vertebrates merge these activities, which are carried out by two independent assemblies in yeast.

Histone acetylation regulates most DNA transactions and is dynamically controlled by highly conserved enzymes. The only essential histone acetyltransferase (HAT) in yeast, Esa1, is part of the 1-MDa NuA4 complex, which plays pivotal roles in both transcription and DNA-damage repair. NuA4 has the unique capacity to acetylate histone targets located several nucleosomes away from its recruitment site. Neither the molecular mechanism of this activity nor its physiological importance are known. Here we report the structure of the Pichia pastoris NuA4 complex, with its core resolved at 3.4-Å resolution. Three subunits, Epl1, Eaf1 and Swc4, intertwine to form a stable platform that coordinates all other modules. The HAT module is firmly anchored into the core while retaining the ability to stretch out over a long distance. We provide structural, biochemical and genetic evidence that an unfolded linker region of the Epl1 subunit is critical for this long-range activity. Specifically, shortening the Epl1 linker causes severe growth defects and reduced H4 acetylation levels over broad chromatin regions in fission yeast. Our work lays the foundations for a mechanistic understanding of NuA4’s regulatory role and elucidates how its essential long-range activity is attained. Here, the authors use cryo-EM, biochemical and yeast assays of the HAT NuA4–Tip60 to reveal its mechanism of acetylating distant nucleosomes through the Epl1 linker establishing long-range chromatin interactions.