Highlights d The g-tubulin ring complex (gTuRC) nucleates microtubules and caps their minus ends d Microtubule nucleation from purified gTuRC is highly cooperative, yet inefficient d A partly open, asymmetric structure of gTuRC explains inefficient nucleation d Actin and MZT2 stabilize the closed part of the gTuRC structure

The exosome plays an important role in RNA degradation and processing. In archaea, three Rrp41:Rrp42 heterodimers assemble into a barrel like structure that contains a narrow RNA entrance pore and a lumen that contains three active sites. Here, we demonstrate that this quaternary structure of the exosome is important for efficient RNA degradation. We find that the entrance pore of the barrel is required for nM substrate affinity. This strong interaction is crucial for processive substrate degradation and prevents premature release of the RNA from the enzyme. Using methyl TROSY NMR techniques, we establish that the 3′ end of the substrate remains highly flexible inside the lumen. As a result, the RNA jumps between the three active sites that all equally participate in substrate degradation. The RNA jumping rate is, however, much faster than the cleavage rate, indicating that not all active site:substrate encounters result in catalysis. Enzymatic turnover therefore benefits from the confinement of the active sites and substrate in the lumen, which ensures that the RNA is at all times bound to one of the active sites. The evolution of the exosome into a hexameric complex and the optimization of its catalytic efficiency were thus likely co-occurring events.

The exosome is a large molecular machine that is involved in RNA degradation and processing. Here, we address how the trimeric Rrp4 cap enhances the activity of the archaeal enzyme complex. Using methyl TROSY NMR methods we identified a 50 Å long RNA binding path on each Rrp4 protomer. We show that the Rrp4 cap can thus recruit three substrates simultaneously, one of which is degraded in the core while two others are positioned for subsequent degradation rounds. The local interaction energy between the substrate and the Rrp4-exosome increases from the periphery of the complex towards the active sites. Importantly, the intrinsic interaction strength between the cap and the substrate is weakened as soon as substrates enter the catalytic barrel, which provides a means to reduce friction during substrate movements towards the active sites. Our data thus reveal a sophisticated exosome–substrate interaction mechanism that enables efficient RNA degradation.

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An ambitious task for human genetics is discovering the genetic basis for disease risks. A key step in this task is predicting whether or not observed genetic variation in coding regions has any impact on protein function. Many bioinformatics tools base predictions on evolutionary conservation, known protein structures, changes in specific amino acid properties, or the local sequence context. Here we examine predictions using a unique first-principles approach based on biophysics to defining the local sequence context. Although not all proteins have well-defined structures, most are intrinsically modular, including disordered proteins. Yet there is currently no straightforward way to incorporate sequence modularity without a corresponding structure for every protein in a dataset. Most sequence-based techniques have relied on moving windows of fixed-width to capture sequence context, blurring the edges between protein modules. We previously developed the blobulation'' approach, which divides the protein into contiguous stretches of residues that do or do not meet a hydrophobicity threshold, for analyzing subtle differences in trajectories of long intrinsically disordered proteins. Here we use the blobulation approach to test for enrichment of disease-associated SNPs within a given protein sequence. Using a dataset of 70,000 SNPs, we find that blobulation yields substantially increased enrichment of disease-associated mutations, relative to a moving window approach. Due to the role of hydrophobicity in stabilizing inter and intraprotein interactions, we further hypothesized that missense variants in hydrophobic blobs, particularly those that change blob boundaries, are more likely to have a functional impact than variants in non-hydrophobic or polar blobs; this hypothesis was confirmed. Moreover, this effect increases with blob length and with the hydrophobicity threshold, such that mutations in long, highly hydrophobic blobs were more than 3.4 times as likely to be disease-associated.

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Pol epsilon is a tetrameric assembly that plays distinct roles during eukaryotic chromosome replication. It catalyses leading strand DNA synthesis; yet this function is dispensable for viability. Its non-catalytic domains instead play an essential role in the assembly of the active replicative helicase and origin activation, while non-essential histone-fold subunits serve a critical function in parental histone redeposition onto newly synthesised DNA. Furthermore, Pol epsilon plays a structural role in linking the RFC–Ctf18 clamp loader to the replisome, supporting processive DNA synthesis, DNA damage response signalling as well as sister chromatid cohesion. In this minireview, we discuss recent biochemical and structural work that begins to explain various aspects of eukaryotic chromosome replication, with a focus on the multiple roles of Pol epsilon in this process.

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