Cyclic GMP-AMP synthase (cGAS) recognizes cytosolic foreign or damaged DNA to activate the innate immune response to infection, inflammatory diseases, and cancer. In contrast, cGAS reactivity against self-DNA in the nucleus is suppressed by chromatin tethering. We report a 3.3-angstrom-resolution cryo-electron microscopy structure of cGAS in complex with the nucleosome core particle. The structure reveals that cGAS employs two conserved arginines to anchor to the nucleosome acidic patch. The nucleosome binding interface exclusively occupies the strong dsDNA binding surface on cGAS and sterically prevents cGAS from oligomerizing into the functionally active 2:2 cGAS-dsDNA state. These findings provide a structural basis for how cGAS maintains an inhibited state in the nucleus and further exemplify the role of the nucleosome in regulating diverse nuclear protein functions.
Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg 2+ ion concentrations are low, K + concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg 2+ (0.5-2 mM) and K + (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperaturedependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.
The protein kinase PKR is an essential component of the innate immune response. In the presence of dsRNA, PKR is autophosphorylated, which enables it to phosphorylate its substrate, eIF2α, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5′-UTR of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and non-canonical features of domain II cumulate to a total of ∼33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV non-structural protein NS5A to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.
Long-range effects, such as allostery, have evolved in proteins as a means of regulating function via communication between distal sites. An NMR-based perturbation mapping approach was used to more completely probe the dynamic response of the core mutation V54A in the protein eglin c by monitoring changes in ps-ns aromatic side-chain dynamics and H/D exchange stabilities. Previous side-chain dynamics studies on this mutant were limited to methyl-bearing residues, most of which were found to rigidify on the ps-ns timescale in the form of a contiguous 'network'. Here, high precision 13 C relaxation data from 13 aromatic side chains were acquired by applying canonical relaxation experiments to a newly-developed carbon labeling scheme [Teilum K. et al. (2006) J. Am. Chem. Soc. 128, 2506-2507. The fitting of model-free parameters yielded S 2 variability which is intermediate with respect to backbone and methyl-bearing side chain variability and τ e values that are approximately one nanosecond. Inclusion of the aromatic dynamic response results in an expanded network of dynamically coupled residues, with some aromatics showing increases in flexibility, which partially offsets the rigidification in methyl side chains. Using amide hydrogen exchange, dynamic propagation on a slower timescale was probed in response to the V54A perturbation. Surprisingly, regional stabilization (slowed exchange) 10-12 angstroms from the site of mutation was observed despite a global destabilization of 1.5 kcal·mol −1 . Furthermore, this unlikely pocket of stabilized residues co-localizes with increases in aromatic flexibility on the faster timescale. Because the converse is also true (destabilized residues co-localize with rigidification on the fast timescale), a plausible entropy-driven mechanism is discussed for relating co-localization of opposing dynamic trends on vastly different timescales. KeywordsNMR; dynamics; relaxation; aromatic; hydrogen exchange; eglin c; V54AMotional flexibility, or dynamics, is essential for protein stability and function (2-6). Recently, native-state protein dynamics have been thought to be crucial for mediating allosteric signaling, or stated more generally, long-range communication (7)(8)(9)(10)(11)(12)(13)(14). One of the major challenges is the experimental characterization of dynamic processes relevant to intra- † This research was supported by NIH Grant GM066009 * To whom correspondence should be addressed: University of North Carolina, School of Pharmacy, Beard Hall, CB# 7360, Chapel Hill, NC 27599-7360. drewlee@unc.edu. Phone: (919) 843-5150. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 23. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript molecular signal transduction. Towards this goal, NMR spectroscopy is uniquely suited to non-invasively characterize both structure and dynamics in a site-specific manner. Mapping flexibility by NMR is therefore an important application for understanding signaling in prote...
It is now widely recognized that dynamics are important to consider for understanding allosteric protein function. However, dynamics occur over a wide range of timescales, and how these different motions relate to one another is not well understood. Here, we report an NMR relaxation study of dynamics over multiple timescales at both backbone and side-chain sites upon an allosteric response to phosphorylation. The response regulator, Escherichia coli CheY, allosterically responds to phosphorylation with a change in dynamics on both the μs-ms timescale and ps-ns timescale. We observe an apparent decrease and redistribution of μs-ms dynamics upon phosphorylation (and accompanying Mg2+ saturation) of CheY. Additionally, methyl groups with the largest changes in ps-ns dynamics localize to the regions of conformational change measured by μs-ms dynamics. The limited spread of changes in ps-ns dynamics suggests a distinct relationship between motions on the μs-ms and ps-ns timescales in CheY. The allosteric mechanism utilized by CheY highlights the diversity of roles dynamics play in protein function.
MicroRNAs are evolutionarily conserved small, non-coding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, we reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site. We show that the alternative excited conformation transiently sequesters the bulged adenine into a non-canonical protonated A + -G mismatch, conferring a two-fold enhancement in Dicer processing over its ground conformational state. These results indicate that microRNA maturation efficiency may be encoded in the intrinsic dynamic ensemble of primary and precursor microRNAs, providing potential means of regulating microRNA biogenesis in response to environmental and cellular stimuli. Page 3 MicroRNAs (miRNAs) are highly conserved, small noncoding RNAs that regulate more than 60% of protein coding genes at the post-transcriptional level 1-5 . Most miRNAs are initially transcribed by RNA polymerase II as introns of protein-coding genes or from independent coding genes into long primary transcripts (pri-miRNAs) that feature 5'-end 7-methylguanosine caps and 3'-end poly-A tails 6,7 . In the canonical biogenesis pathway, pri-miRNAs are subsequently processed into ~70 nucleotide precursor hairpins (pre-miRNAs) by the Microprocessor complex, consisting of one RNase III family enzyme, Drosha, and two DiGeorge critical region 8 proteins (DGCR8) 8,9 . Pre-miRNAs are then exported from the nucleus to the cytoplasm by Exportin-5 (Ref. 10) and further processed into ~20 base-pair miRNA/miRNA* duplexes by another RNase III family enzyme, Dicer, in complex with transactivation-responsive RNA binding protein (TRBP) 11,12 . The resulting single-stranded mature miRNA is incorporated into the miRNA-inducing silencing complex (miRISC), which regulates protein expression by repressing translation, promoting deadenylation, and/or cleaving target mRNA 13 .Due to their essential regulatory roles, miRNA biogenesis is tightly regulated to ensure proper gene expression 3-5 , and abnormal miRNA regulation has often been associated with cancer, neurological disorders, cardiovascular diseases and others 14,15 .Remarkably, despite sharing the same set of enzymes in the canonical biogenesis pathway, individual miRNAs exhibit cell-type and cell-state specific expressions. Even those clustered on the same primary transcript can be differentially processed in a tissue-specific manner 3-5 . Over the past decade, it has been shown that specific sequences and structures of primary and precursor miRNAs can be recognized by processing machineries and protein factors for regulation [16][17][18][19][20][21][22][23][24][25][26] . For example, pri-miRNAs
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