Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis

Amplification of short interfering RNA (siRNAs) via RNA dependent RNA Polymerases (RdRPs) is of fundamental importance in RNA silencing. In plants, silencing by microRNAs (miRNAs) generally does not lead to engagement of RdRPs, in part thanks to an as yet poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that mutation of the cytoplasmic exosome subunit RRP45B results in siRNA production very similar to what is observed in ski2 mutants. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that miRNA-induced illicit secondary siRNA production correlates with miRNA levels rather than accumulation of stable 5'-cleavage fragments. We propose that stalled RNA-induced Silencing Complex (RISC) and ribosomes, but not stable target mRNA cleavage fragments released from RISC, trigger secondary siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.

A Universal Allosteric Mechanism for G Protein Activation

SUMMARYG proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote GTP binding and the dissociation of Gα from the Gβγ subunits. Structural studies have revealed the molecular basis for subunit association with receptors, RGS proteins and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We used cell signaling assays, MD simulations, biochemistry and structural analysis to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This “G-R-E motif” secures GTP and, through an allosteric link, discharges the Gβγ dimer. Replacement of network residues prevents subunit dissociation, regardless of agonist or GTP binding. These findings reveal the molecular basis for the final committed step of G protein activation.HIGHLIGHTSReceptors promote GTP-GDP exchange and dissociation of G protein α and βγ subunitsWe find an allosteric network linking the γ phosphate of GTP with release of GβγThe network consists of a conserved Gly-Arg-Glu “activation triad”Triad mutations prevent subunit dissociation, regardless of agonist or GTP bindingTriad mutations are responsible for human endocrine and neurological disorders

The structural basis for inhibition of ribosomal translocation by viomycin

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.

Potassium Glutamate and Glycine Betaine Induce Self-Assembly of Sliding Clamps into Higher Order Oligomers

ABSTRACTSliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli β-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of β is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the S. cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to β. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.

Swimming regulations for protein kinase A catalytic subunit

cAMP-dependent protein kinase (PKA) plays a central role in important biological processes including synaptic plasticity and sympathetic stimulation of the heart. Elevations of cAMP trigger release of PKA catalytic (C) subunits from PKA holoenzymes, thereby coupling cAMP to protein phosphorylation. Uncontrolled C subunit activity, such as occurs in genetic disorders in which regulatory subunits are depleted, is pathological. Anchoring proteins that associate with PKA regulatory subunits are important for localising PKA activity in cells. However, anchoring does not directly explain how unrestrained ‘free swimming' of C subunits is avoided following C subunit release. In this review, I discuss new mechanisms that have been posited to account for this old problem. One straightforward explanation is that cAMP does not trigger C subunit dissociation but instead activates intact PKA holoenzymes whose activity is restrained through anchoring. A comprehensive comparison of observations for and against cAMP-activation of intact PKA holoenzymes does not lend credence to this mechanism. Recent measurements have revealed that PKA regulatory subunits are expressed at very high concentrations, and in large molar excess relative to C subunits. I discuss the implications of these skewed PKA subunit concentrations, before considering how phosphorylation of type II regulatory subunits and myristylation of C subunits are likely to contribute to controlling C subunit diffusion and recapture in cells. Finally, I speculate on future research directions that may be pursued on the basis of these emerging mechanisms.

Topology and cooperative stability: the two master regulators of protein half-life in the cell

In a quest for finding additional structural constraints, apart from disordered segments, regulating protein half-life in the cell (and during evolution), here we recognize and assess the influence of native topology of biological proteins and their sequestration into multimeric complexes. Native topology acts as a molecular marker of protein’s mechanical resistance and consequently captures their half-life variations on genome-scale, irrespective of the enormous sequence, structural and functional diversity of the proteins. Cooperative stability (slower degradation upon sequestration into complexes) is a master regulator of oligomeric protein half-life that involves at least three mechanisms. (i) Association with multiple complexes results longer protein half-life; (ii) hierarchy of complex self-assembly involves short-living proteins binding late in the assembly order and (iii) binding with larger buried surface area leads to slower subunit dissociation and thereby longer half-life. Altered half-lives of paralog proteins refer to their structural divergence and oligomerization with non-identical set of complexes.