Architecture and flexibility of native kinetochores revealed by structural studies utilizing a thermophilic yeast.

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from Kluyveromyces marxianus and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

Computed structures of core eukaryotic protein complexes.

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.

Dynamic Interplay of RNA and Protein in the Human Immunodeficiency Virus-1 Reverse Transcription Initiation Complex.

The initiation of reverse transcription in human immunodeficiency virus-1 is a key early step in the virus replication cycle. During this process, the viral enzyme reverse transcriptase (RT) copies the single-stranded viral RNA (vRNA) genome into double-stranded DNA using human tRNALys3 as a primer for initiation. The tRNA primer and vRNA genome contain several complementary sequences that are important for regulating reverse transcription initiation kinetics. Using single-molecule Förster resonance energy transfer spectroscopy, we demonstrate that the vRNA-tRNA initiation complex is conformationally heterogeneous and dynamic in the absence of RT. As shown previously, nucleic acid-RT interaction is characterized by rapid dissociation constants. We show that extension of the vRNA-tRNA primer binding site helix from 18 base pairs to 22 base pairs stabilizes RT binding to the complex and that the tRNA 5' end has a role in modulating RT binding. RT occupancy on the complex stabilizes helix 1 formation and reduces global structural heterogeneity. The stabilization of helix 1 upon RT binding may serve to destabilize helix 2, the first pause site for RT during initiation, during later steps of reverse transcription initiation.