The recently identified plant photoreceptor UVR8 triggers regulatory changes in gene expression in response to ultraviolet-B (UV-B) light via an unknown mechanism. Here, crystallographic and solution structures of the UVR8 homodimer, together with mutagenesis and far-UV circular dichroism spectroscopy, reveal its mechanisms for UV-B perception and signal transduction. β-propeller subunits form a remarkable, tryptophan-dominated, dimer interface stitched together by a complex salt-bridge network. Salt-bridging arginines flank the excitonically coupled cross-dimer tryptophan “pyramid” responsible for UV-B sensing. Photoreception reversibly disrupts salt bridges, triggering dimer dissociation and signal initiation. Mutation of a single tryptophan to phenylalanine re-tunes the photoreceptor to detect UV-C wavelengths. Our analyses establish how UVR8 functions as a photoreceptor without a prosthetic chromophore to promote plant development and survival in sunlight.

RNA interference is a powerful mechanism of gene silencing that underlies many aspects of eukaryotic biology. On the molecular level, RNA interference is mediated by a family of ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs), which can be programmed to target virtually any nucleic acid sequence for silencing. The ability of RISC to locate target RNAs has been co-opted by evolution many times to generate a broad spectrum of gene-silencing pathways. Here ,we review the fundamental biochemical and biophysical properties of RISC that facilitate gene targeting and describe the various mechanisms of gene silencing known to exploit RISC activity. RISC2 is a generic term for a family of heterogeneous molecular complexes that can be programmed to target almost any gene for silencing. In general, RISC programming is triggered by the appearance of dsRNA in the cytoplasm of a eukaryotic cell (Fig. 1). The dsRNA is processed into small regulatory RNAs (20 -30 nucleotides in length) that assemble into RISC and guide the complex to complementary RNA targets through base-pairing interactions. Once programmed with a small RNA, RISC can silence targeted genes by one of several distinct mechanisms, working at (a) the level of protein synthesis through repression of translation, (b) the transcript level through mRNA degradation, or (c) the level of the genome itself through the formation of heterochromatin or by DNA elimination.Although the mechanisms used to control gene expression by RISC are quite diverse, two central themes are common to all. First, at its core, every RISC contains a member of the Argonaute protein family that binds to the small regulatory RNA. Second, in every RISC, the small regulatory RNA functions as a guide that leads RISC to its target through Watson-Crick base pairing with cognate RNA transcripts. The role of the Argonaute protein is to bind the small RNA and position it in a conformation that facilitates target recognition. Argonaute proteins can either cleave target RNAs directly or recruit other gene-silencing proteins to identified targets. Here, we review how Argonaute proteins use small RNAs to recognize target transcripts. We also examine how recruitment of different types of Argonaute and Argonaute-associated proteins produce distinct RISCs, which then dictate the mechanism of gene regulation. RISC and Small RNA NomenclatureAs in many fields of biology, the nomenclature commonly used to describe RNAi and RISC is not completely rational or intuitive. The small regulatory RNAs that guide RISC have been given a variety of similar sounding names. These include siRNA, miRNA, piRNA, rasiRNA, tasiRNA, tncRNA, hcRNA, and scnRNA. These classifications are generally based on either the biosynthetic pathway of the small RNA or the type of RISC in which the RNA is found (for a detailed review, see Ref.

Background: iLOV is a fluorescent flavoprotein engineered from the plant blue light receptor phototropin. Results: Structures reveal altered protein-chromophore interactions within the flavin-binding cavity of iLOV when compared with its progenitors. Directed evolution further anchored the chromophore to increase iLOV photostability by an order of magnitude. Conclusion: Improving iLOV photostability by constraining its fluorophore establishes a framework for fine-tuning fluorescence. Significance: Enhanced photostability increases iLOV utility as an oxygen-independent fluorescent reporter.

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu 2+ binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies. Lou Gehrig's disease | small-angle X-ray scattering | protein aggregation | protein conformation | ESR spectroscopy A LS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2-4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11,12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14,15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3,(16)(17)(18)(19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework dest...