INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the ...histone variants H2A and H2A.Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our analysis establishes a structural and functional framework for this family of large remodelers.
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•EM structure of INO80 complex shows a distinct shape and conformational dynamics•Crosslinking and mass spectrometry reveal the subunit interaction architecture•Integrative structural biology allows localization of modules and nucleosome•The Rvb1/2 dodecamer and Arp5, Nhp10, and Arp8 modules flank the central Snf2 ATPase
The architecture of the INO80, revealed through a combination of electron microscopy, crosslinking, and mass spectrometry, shows a concave binding surface that cradles nucleosomes in a recognition mode that is distinct from other families of chromatin remodelers.
MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the ...GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago–GW182 interaction. Our data further indicate that the Ago–GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa.
Protein transport into the nucleus is mediated by transport receptors. Import of highly charged proteins, such as histone H1 and ribosomal proteins, requires a dimer of two transport receptors. In ...this study, we determined the cryo-EM structure of the Imp7:Impβ:H1.0 complex, showing that the two importins form a cradle that accommodates the linker histone. The H1.0 globular domain is bound to Impβ, whereas the acidic loops of Impβ and Imp7 chaperone the positively charged C-terminal tail. Although it remains disordered, the H1 tail serves as a zipper that closes and stabilizes the structure through transient non-specific interactions with importins. Moreover, we found that the GGxxF and FxFG motifs in the Imp7 C-terminal tail are essential for Imp7:Impβ dimerization and H1 import, resembling importin interaction with nucleoporins, which, in turn, promote complex disassembly. The architecture of many other complexes might be similarly defined by rapidly exchanging electrostatic interactions mediated by disordered regions.
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•Importin 7 and Importin β form a cradle to chaperone and transport histone H1•Transient and non-specific electrostatic interactions form and shape the complex•The H1 tail serves as a zipper that closes and stabilizes Imp7:Impβ:H1•FxFG motifs in nucleoporins facilitate RanGTP-dependent disassembly of the complex
Ivic et al. determined the cryo-EM structure of the Imp7:Impβ:H1 complex and show that transient and non-specific charged interactions form and shape the complex. They also found that the FxFG motif in Imp7 is essential for complex formation and that nucleoporins promote complex disassembly.
Capping is the first step in pre-mRNA processing, and the resulting 5′-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by ...RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of the capping enzyme (CE) to transcribing, phosphorylated yeast Pol II (Pol IIp) requires nascent RNA with an unprocessed 5′-triphosphate end. The transcribing Pol IIp-CE complex catalyzes the first two steps of capping, and its analysis by mass spectrometry, cryo-electron microscopy, and protein crosslinking revealed the molecular basis for transcription-coupled pre-mRNA capping. CE docks to the Pol II wall and spans the end of the RNA exit tunnel to position the CE active sites for sequential binding of the exiting RNA 5′ end. Thus, the RNA 5′ end triggers its own capping when it emerges from Pol II, to ensure seamless RNA protection from 5′-exonucleases during early transcription.
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•Stable binding of capping enzyme to Pol II requires RNA with a 5′-triphosphate•Architecture of Pol-II-capping enzyme complex is derived from cryo-EM and crosslinking•Capping enzyme binds at RNA exit tunnel, explaining seamless RNA protection
Co-transcriptional capping is the first step in pre-mRNA processing and required for mRNA stability and translation. Martinez-Rucobo et al. show that the capping enzyme binds around the RNA exit tunnel of RNA polymerase II to ensure seamless RNA protection.
Polycomb Repressive Complex 2 (PRC2) is essential for gene silencing, establishing transcriptional repression of specific genes by tri-methylating Lysine 27 of histone H3, a process mediated by ...cofactors such as AEBP2. In spite of its biological importance, little is known about PRC2 architecture and subunit organization. Here, we present the first three-dimensional electron microscopy structure of the human PRC2 complex bound to its cofactor AEBP2. Using a novel internal protein tagging-method, in combination with isotopic chemical cross-linking and mass spectrometry, we have localized all the PRC2 subunits and their functional domains and generated a detailed map of interactions. The position and stabilization effect of AEBP2 suggests an allosteric role of this cofactor in regulating gene silencing. Regions in PRC2 that interact with modified histone tails are localized near the methyltransferase site, suggesting a molecular mechanism for the chromatin-based regulation of PRC2 activity.DOI:http://dx.doi.org/10.7554/eLife.00005.001.
Chromosome segregation depends on sister chromatid cohesion mediated by cohesin. The cohesin subunits Smc1, Smc3, and Scc1 form tripartite rings that are thought to open at distinct sites to allow ...entry and exit of DNA. However, direct evidence for the existence of open forms of cohesin is lacking. We found that cohesin's proposed DNA exit gate is formed by interactions between Scc1 and the coiled-coil region of Smc3. Mutation of this interface abolished cohesin's ability to stably associate with chromatin and to mediate cohesion. Electron microscopy revealed that weakening of the Smc3-Scc1 interface resulted in opening of cohesin rings, as did proteolytic cleavage of Scc1. These open forms may resemble intermediate states of cohesin normally generated by the release factor Wapl and the protease separase, respectively.
Kinetochores are megadalton-sized protein complexes that mediate chromosome-microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is ...poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1(CENP-U) and Okp1(CENP-Q) form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1-Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.
The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that 'axis ...core proteins' from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify 'closure motifs' in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control.
Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer ...kinetochore, assembling into 3 MDa‐sized microtubule‐embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi‐orientation. We show that Dam1c and the general microtubule plus end‐associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c‐Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c‐Bim1 binding by relieving an intramolecular inhibition of the Dam1 C‐terminus. In addition, Bim1 recruits Bik1/CLIP‐170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end‐on attachments are formed during the process of attachment error correction.
SYNOPSIS
Kinetochore‐microtubule attachment for budding yeast chromosome segregation requires ring formation of the Dam1 complex (Dam1c). A combination of genetic, biochemical and structural approaches reveals how association of microtubule plus‐end associated proteins triggers formation of Dam1c rings.
Plus end‐binding protein Bim1 binds and crosslinks the protrusion domains of Dam1c.
Interaction with Bim1 is required for efficient Dam1c kinetochore loading in metaphase and for timely mitotic progression.
Mps1 kinase activity promotes Dam1c‐Bim1 complex formation.
Bim1 cooperates with CLIP‐170 homolog Bik1 to induce Dam1c ring assembly in vitro independent of microtubules.
The Dam1c‐Bim1‐Bik1 complex might represent an early configuration during end‐on attachment formation.
A combination of genetic, biochemical and structural approaches reveals how phosphorylation‐regulated association of microtubule plus‐end associated proteins facilitates formation of complexes for end‐on spindle attachment.
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