Allostery is well documented for proteins but less recognized for DNA-protein interactions. Here we report that specific binding of a protein on DNA is substantially stabilized or destabilized by another protein bound nearby. The ternary complex's free energy oscillates as a function of the separation between the two proteins with a periodicity of ~10 base pairs, the helical pitch of B-form DNA, and a decay length of ~15 base pairs. The binding affinity of a protein near a DNA hairpin is similarly dependent on their separation, which—together with molecular dynamics simulations—suggests that deformation of the double-helical structure is the origin of DNA allostery. The physiological relevance of this phenomenon is illustrated by its effect on gene expression in live bacteria and on a transcription factor's affinity near nucleosomes.

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) mediates innate immune responses against invading pathogens, or against self-dsDNA, which causes autoimmune disorders. Upon nonspecific binding of cytosolic B-form DNA, cGAS synthesizes the second messenger 2'3'-cGAMP and triggers STING-dependent signaling to produce type I IFNs. The cGAS comprises less-conserved N-terminal residues and highly conserved nucleotidyltransferase/Mab21 domains. The function and structure of the well-conserved domains have been extensively studied, whereas the physiological function of the N-terminal domain of cGAS is largely uncharacterized. In this study we used a single-molecule technique combined with traditional biochemical and cellular assays to demonstrate that binding of nonspecific dsDNA by the N-terminal domain of cGAS promotes its activation. We have observed that the N terminus of human cGAS (cGAS-N160) undergoes secondary structural change upon dsDNA binding in solution. Furthermore, we showed that the cGAS-N160 helps full lengthcGAS to expand the binding range on λDNA and facilitates its binding efficiency to dsDNA compared with cGAS without the 160 N-terminal residues (cGAS-d160). More importantly, cGAS-N160 endows full lengthcGAS relatively higher enzyme activity and stronger activation of STING/IRF3-mediated cytosolic DNA signaling. These findings strongly indicate that the N-terminal domain of cGAS plays an important role in enhancing its function.

The mean lifetimes r of the Ca + 4p 2 Pin and 2 P\n levels have been measured to ==0.3% precision using a variant of the collinear laser-beam-ion-beam spectroscopy technique. Like previously measured precision lifetimes of light alkali-metal-like systems, these data fall significantly higher than calculations by the most recent multiconfiguration Hartree-Fock and relativistic many-body perturbation theories. Discrepancies between experiment and theory for alkali-metal-like systems have implications for the interpretation of parity-violation research.PACS numbers: 32.70.Fw, 31.30Jv, 42.55.Px, 42.62.Fi There is a current demand for precision (better than 1%) measurements of the lifetimes of electric dipole transitions of atoms or ions with a single electron outside a closed electron shell [1]. This arises from persistent attempts to improve the accuracy of relativistic many-body perturbation theory (MBPT) and multiconfiguration Dirac-Fock (MCDF) or Hartree-Fock (MCHF) calculations to this level of precision and beyond. Theoretical accuracy in transition matrix elements is required in conjunction with experimental precision (currently at the 2% level) in interference in transition amplitudes, to obtain the weak coupling constants in the analysis of atomic physics measurements [2] of parity violation in heavy, many-electron alkali-metal atoms such as Cs.During the last decade, discrepancies at the 1% level have been noted between the most precise experimental lifetimes of electric dipole decays of levels of light alkali metals, and ab initio theoretical lifetime calculations [3]. The measurements fall higher than the theory typically by > 5 standard deviations ( 3/2 level is r (Ca + ,4p 2^3/2) =6.924(0.019) ns while the corresponding measurement for the 2 P\/2 level is r (Ca + , 4p 2 / > i/2> =7.098(0.020) ns. Our results agree with, but are 3 times more precise than, the crossed laser-ion-beam data of Gosselin, Pinnington, and Ansbacher [6]. Our lifetime data fall higher than all of the ab initio theoretical calculations by about 2% (6a) or more, and 0.75% (2.5cr) or more higher than all semiempirical calculations. The disagreement between the theoretical and experimental results for Ca + is in accord with the earlier trend [3] for the few-electron alkali-metal-like systems, possibly signaling fundamental problems.Lifetime determinations based on laser excitation of fast beam atoms or ions are known for their accuracy and precision, since a single level is excited by the laser, and particle velocities can be well establ...

DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.

Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244 − CXCR6 − ), C1 (CD244 − CXCR6 + ), or C2 (CD244 + CXCR6 + ) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell–like features, whereas C1 iNKT cells showed more T cell–like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244 + CXCR6 + iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell–like properties distinct from conventional tissue-resident iNKT cells.

Tumors are complex ecosystems in which heterogeneous cancer cells interact with their microenvironment composed of diverse immune, endothelial, and stromal cells. Cancer biology had been studied using bulk genomic and gene expression profiling, which however mask the cellular diversity and average the variability among individual molecular programs. Recent advances in single-cell transcriptomic sequencing have enabled a detailed dissection of tumor ecosystems and promoted our understanding of tumorigenesis at single-cell resolution. In the present review, we discuss the main topics of recent cancer studies that have implemented single-cell RNA sequencing (scRNA-seq). To study cancer cells, scRNA-seq has provided novel insights into the cancer stem-cell model, treatment resistance, and cancer metastasis. To study the tumor microenvironment, scRNA-seq has portrayed the diverse cell types and complex cellular states of both immune and non-immune cells interacting with cancer cells, with the promise to discover novel targets for future immunotherapy.

In this work, we developed a method to systematically study the sequence preference of mRNAs during translation initiation. Traditionally, the dynamic process of translation initiation has been studied at the single molecule level with limited sequencing possibility. Using deep sequencing techniques, we identified the sequence preference at different stages of the initiation complexes. Our results provide a comprehensive and dynamic view of the initiation elements in the translation initiation region (TIR), including the S1 binding sequence, the Shine-Dalgarno (SD)/anti-SD interaction and the second codon, at the equilibrium of different initiation complexes. Moreover, our experiments reveal the conformational changes and regional dynamics throughout the dynamic process of mRNA recruitment.

Metastable multiply charged argon ions produced in, and extracted from, an electron cyclotron resonance ion source were captured from the beam into an electrostatic (Kingdon) ion trap by rapidly pulsing the potential of the central wire relative to the cylinder. The ions were selected on a charge-to-mass ratio basis before capture. Photons emitted in magnetic-dipole and electric-quadrupole transitions from levels with lifetimes exceeding 5 ms were selected by wavelength and recorded vs ion storage time in the trap. Also, ions were counted after ejection following a preselected storage time in the trap, using a mi-