Biology subjects

Chait, B. T.

Publications and source records attributed to Chait, B. T..

26 records · Page 2Linked to original sources

bioRxiv2020

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

The formation of cellular microtubule networks is regulated by the{gamma} -tubulin ring complex ({gamma}-TuRC). This [~]2.3 MDa assembly of >31 proteins includes{gamma} -tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge". The challenge of reconstituting the{gamma} -TuRC has limited dissections of its assembly and function. Here, we report a complete biochemical reconstitution of the human{gamma} -TuRC ({gamma}-TuRC-GFP), a [~]35S complex that nucleates microtubules in vitro. We extend our approach to generate a stable subcomplex,{gamma} -TuRCmini-GFP, which lacks MZT1 and actin. Using mutagenesis, we show that{gamma} -TuRCmini-GFP nucleates microtubules in a guanine nucleotide-dependent manner and proceeds with similar kinetics as reported for native{gamma} -TuRCs. Electron microscopy reveals that{gamma} -TuRC-GFP resembles the native{gamma} -TuRC architecture, while{gamma} -TuRCmini-GFP adopts a partial cone shape presenting only 8-10{gamma} -tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the{gamma} -TuRC.

biochemistry↗

bioRxiv2020

Structural basis for transcription complex disruption by the Mfd translocase

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (EC). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

biophysics↗

bioRxiv2020

Affinity proteomic dissection of the human nuclear cap-binding-complex interactome

A 5, 7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). The CBC has come under renewed investigative interest in recent years due to its participation in RNA-fate decisions via interactions with RNA productive factors as well as with adapters of the degradative RNA exosome - including the proteins SRRT (a.k.a. ARS2) and ZC3H18, and macromolecular assemblies such as the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. A novel cap-binding protein, NCBP3, was recently proposed to form an alternative, non-canonical CBC together with NCBP1, and to interact with the canonical CBC along with the protein SRRT. The theme of post-transcriptional RNA fate, and how it relates to co-transcriptional ribonucleoprotein assembly is abundant with complicated, ambiguous, and likely incomplete models. In an effort to clarify the compositions of NCBP1-, 2-, and 3-related macromolecular assemblies, including their intersections and differences, we have applied an affinity capture-based interactome screening approach, where the experimental design and data processing have been modified and updated to identify interactome differences between targets under a range of experimental conditions, in the context of label-free quantitative mass spectrometry. This study generated a comprehensive view of NCBP-protein interactions in the ribonucleoprotein context and demonstrates the potential of our approach to benefit the interpretation of complex biological pathways.

biochemistry↗

bioRxiv2019

Asymmetric molecular architecture of the human γ-tubulin ring complex

The{gamma} -tubulin ring complex ({gamma}-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation 1-4. Metazoan{gamma} -TuRCs isolate as [~]2 MDa complexes containing the conserved proteins{gamma} -tubulin, GCP2 and GCP3, as well as the expanded subunits GCP4, GCP5, and GCP6 3,5,6. However, in current structural models,{gamma} -TuRCs assemble solely from subcomplexes of{gamma} -tubulin, GCP2 and GCP3 7. The role of the metazoan-specific subunits in{gamma} -TuRC assembly and architecture are not currently known, due to a lack of high resolution structural data for the native complex. Here, we present a cryo-EM structure of the native human{gamma} -TuRC at 3.8[A] resolution. Our reconstruction reveals an asymmetric, single helical-turn and cone-shaped structure built from at least 34 polypeptides. Pseudo-atomic models indicate that GCP4, GCP5 and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes and are distal to the{gamma} -TuRC \"seam\". Evolutionary expansion in metazoan-specific subunits diversifies the{gamma} -TuRC by introducing large (>100,000 [A]2) surfaces that could interact with different regulatory factors. We also identify an unanticipated structural bridge that includes an actin-like protein and spans the{gamma} -TuRC lumen. Despite its asymmetric composition and architecture, the human{gamma} -TuRC arranges{gamma} -tubulins into a helical geometry poised to nucleate microtubules. The observed compositional complexity of the{gamma} -TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g. within biological condensates 8 or alongside existing filaments 9.

biochemistry↗

bioRxiv2018

Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations

Protein-protein interactions are central to biological processes and the methods to thoroughly characterize them are of great interest. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions involved in nucleocytoplasmic transport as a model system, we examined the utility of three surface-based methods in characterizing rapid interactions involving intrinsically disordered proteins: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the existence of previously overlooked mass transport limitations was revealed. Additional experiments with a loss-of-interaction mutant variant demonstrated the existence of additional physical events and an uncharacterized binding mode. These results suggest the binding events that take place on the surface are more complex than initially assumed, prompting a need for re-interpretation of previous data.

biophysics↗

bioRxiv2017

Modular assembly of the nucleolar large subunit processome

Early co-transcriptional events of eukaryotic ribosome assembly result in the formation of the small and large subunit processomes. We have determined cryo-EM reconstructions of the nucleolar large subunit processome in different conformational states at resolutions up to 3.4 [A]ngstroms. These structures reveal how steric hindrance and molecular mimicry are used to prevent premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Mutually exclusive conformations of these particles suggest that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly.

biochemistry↗

bioRxiv2017

The complete structure of the small subunit processome

The small subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small subunit processome at an overall resolution of 3.8 [A], which provides an essentially complete atomic model of this assembly. In this nucleolar superstructure, 51 ribosome assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry, steric hindrance as well as protein-and RNA-mediated RNA remodeling are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.

biochemistry↗

bioRxiv2017

Dissection of purified LINE-1 reveals distinct nuclear and cytoplasmic intermediates

1.Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications and quantitative mass spectrometry, we have characterized the proteins associated with distinctive L1 macromolecular complexes. Our findings support the presence of multiple L1-derived retrotransposition intermediates in vivo. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in template-primed reverse transcription.

molecular biology↗