The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanisms: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. By comprehensively analyzing the three dimensional structures of protein-DNA complexes, we show that the binding of arginines to narrow minor grooves is a widely used mode for protein-DNA recognition. This readout mechanism exploits the phenomenon that narrow minor grooves strongly enhance the negative electrostatic potential of the DNA. The nucleosome core particle offers a striking example of this effect. Minor groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings suggest that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA binding specificity.

Specific interactions between proteins and DNA are fundamental to many biological processes. In this review, we provide a revised view of protein-DNA interactions that emphasizes the importance of the three-dimensional structures of both macromolecules. We divide protein-DNA interactions into two categories: those where the protein recognizes the unique chemical signatures of the DNA bases (base readout) and those where the protein recognizes a sequence-dependent DNA shape (shape readout). We further divide base readout into those interactions that occur in the major groove from those that occur in the minor groove. Analogously, the readout of DNA shape is subdivided into global shape recognition, for example when the DNA helix exhibits an overall bend, and local shape recognition, for example when a base pair step is kinked or when a region of the minor groove is narrow. Based on the >1500 structures of protein-DNA complexes now available in the Protein Data Base, we argue that individual DNA binding proteins combine multiple readout mechanisms to achieve DNA binding specificity. Specificity that distinguishes between families frequently involves base readout in the major groove while shape readout is often exploited for higher resolution specificity, to distinguish between members within the same DNA-binding protein family.

It has been known for some time that the double-helix is not a uniform structure but rather exhibits sequence-specific variations that, combined with base-specific intermolecular interactions, offer the possibility of numerous modes of protein-DNA recognition. All-atom simulations have revealed mechanistic insights into the structural and energetic basis of various recognition mechanisms for a number of protein-DNA complexes while coarser grained simulations have begun to provide an understanding of the function of larger assemblies. Molecular simulations have also been applied to the prediction of transcription factor binding sites, while empirical approaches have been developed to predict nucleosome positioning. Studies that combine and integrate experimental, statistical and computational data offer the promise of rapid advances in our understanding of protein-DNA recognition mechanisms.

DNA shape variation and the associated variation in minor groove electrostatic potential are widely exploited by proteins for DNA recognition. Here we show that the hydroxyl radical cleavage pattern is a quantitative measure of DNA backbone solvent accessibility, minor groove width, and minor groove electrostatic potential, at single nucleotide resolution. We introduce maps of DNA shape and electrostatic potential as tools for understanding how proteins recognize binding sites in a genome. These maps reveal periodic structural signals in yeast and Drosophila genomic DNA sequences that are associated with positioned nucleosomes.

Proteins rely on a variety of readout mechanisms to preferentially bind specific DNA sequences. The nucleosome offers a prominent example of a shape readout mechanism where arginines insert into narrow minor groove regions that face the histone core. Here we compare DNA shape and arginine recognition of three nucleosome core particle structures, expanding on our previous study by characterizing two additional structures, one with a different protein sequence and one with a different DNA sequence. The electrostatic potential in the minor groove is shown to be largely independent of the underlying sequence but is, however, dominated by groove geometry. Our results extend and generalize our previous observation that the interaction of arginines with narrow minor grooves plays an important role in stabilizing the deformed DNA in the nucleosome.

BackgroundThe 3′ untranslated regions (UTRs) of mRNAs play a major role in post-transcriptional regulation of gene expression. Selection of transcript cleavage and polyadenylation sites is a dynamic process that produces multiple transcript isoforms for the same gene within and across different cell types. Using LITE-Seq, a new quantitative method to capture transcript 3′ ends expressed in vivo, we have characterized sex- and cell type-specific transcriptome-wide changes in gene expression and 3′UTR diversity in Caenorhabditis elegans germline cells undergoing proliferation and differentiation.ResultsWe show that nearly half of germline transcripts are alternatively polyadenylated, that differential regulation of endogenous 3′UTR variants is common, and that alternative isoforms direct distinct spatiotemporal protein expression patterns in vivo. Dynamic expression profiling also reveals temporal regulation of X-linked gene expression, selective stabilization of transcripts, and strong evidence for a novel developmental program that promotes nucleolar dissolution in oocytes. We show that the RNA-binding protein NCL-1/Brat is a posttranscriptional regulator of numerous ribosome-related transcripts that acts through specific U-rich binding motifs to down-regulate mRNAs encoding ribosomal protein subunits, rRNA processing factors, and tRNA synthetases.ConclusionsThese results highlight the pervasive nature and functional potential of patterned gene and isoform expression during early animal development.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor–ligand interface and receptor–receptor interface; the unique C-terminal receptor–receptor enables higher order structures on the membrane. Human GITR–GITRL has potential to form a hexameric network of membrane complexes, while murine GITR–GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor–receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.

was captured on anti-mouse Fc tips. Mouse Tim-3-human Fc fusion protein was captured on anti-human Fc tips. Captured Tim-3 was first saturated with an antibody (aTim-3.18, aTim-3.22, RMT3-23, or nonblocking anti-mouse aTim-3 control mAb), and then binding of PS liposomes was tested. The observed PS binding signal was normalized to the highest binding response in the assay.