Sharply bent DNA is essential for gene regulation in prokaryotes and is a major feature of eukaryotic nucleosomes and viruses. The explanation normally given for these phenomena is that specific proteins sharply bend DNA by application of large forces, while the DNA follows despite its intrinsic inflexibility. Here we show that DNAs that are 94 bp in length-comparable to sharply looped DNAs in vivo-spontaneously bend into circles. Proteins can enhance the stability of such loops, but the loops occur spontaneously even in naked DNA. Random DNA sequences cyclize 10(2)-10(4) times more easily than predicted from current theories of DNA bending, while DNA sequences that position nucleosomes cyclize up to 10(5) times more easily. These unexpected results establish DNA as an active participant in the formation of looped regulatory complexes in vivo, and they point to a need for new theories of DNA bending.

Gene-regulatory complexes often require that pairs of DNA-bound proteins interact by looping-out short (often Ϸ100-bp) stretches of DNA. The loops can vary in detailed length and sequence and, thus, in total helical twist, which radically alters their geometry. How this variability is accommodated structurally is not known. Here we show that the inherent twistability of 89-to 105-bp DNA circles exceeds theoretical expectation by up to 400-fold. These results can be explained only by greatly enhanced DNA flexibility, not by permanent bends. They invalidate the use of classic theories of flexibility for understanding sharp DNA looping but support predictions of two recent theories. Our findings imply an active role for DNA flexibility in loop formation and suggest that variability in the detailed helical twist of regulatory loops is accommodated naturally by the inherent twistability of the DNA.activation ͉ gene regulation ͉ repression D ouble-stranded DNA in vivo is often sharply distorted away from its classic B-form conformations. In many prokaryotic gene-regulatory complexes, short stretches of DNA, Ϸ100 bp in length, are bent sharply into circular or antiparallel (U-shaped or teardrop-shaped) loops (1, 2). Looping allows synergy between proteins bound at distant DNA sites (3) and decreases the statistical noise in their occupancy (4). DNA looping is also important in eukaryotic regulatory systems (5-7). A striking recent example is the discovery of ligand-dependent DNA looping by the RXR receptor, which may play an important regulatory role at as many as 172 different locations, genome wide, in the mouse (8). In addition, most (Ϸ75-80%) of the length of eukaryotic genomic DNA is sharply bent into nucleosomes (80-bp superhelical loops) (9), which regulate the accessibility and proximity of other DNA-functional sites (10, 11).Prokaryotic and eukaryotic regulatory complexes involving short DNA loops (12-18) place strong constraints on the helical twist of the looped DNA (2). For two DNA-bound proteins to interact when they are separated along the DNA, the proteinbinding sites need to occur on mutually compatible faces of the DNA double helix. This requirement is satisfied by a set of lengths for the intervening DNA that differ from one another by integral multiples of the DNA helical repeat, Ϸ10.5 bp. When the exact length of the intervening DNA in such complexes is suboptimal, the DNA may be under-or overtwisted to allow the protein-protein interaction. The DNA helical twist is altered in other biological systems as well, most notably in the nucleosome, in which the wrapped DNA is under-or overtwisted for most of its length (9).DNA bending and twisting deformations that are required for protein-DNA complex formation come at a cost in free energy, which contributes importantly to the net stabilities and functions of the resulting complexes. For these reasons, the inherent bendability and twistability of DNA itself have been a focus of experimental and theoretical investigation. Classic studies revealed double-st...

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Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.

High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATAbinding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (⌬G°‫؍‬ ؊12.10 ؎ 0.12 kcal͞mol) and TFIIIB-DNA (⌬G°‫؍‬ ؊11.90 ؎ 0.14 kcal͞mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 ؎ 3 min and 95 ؎ 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation. RNA polymerase (pol) III transcribes a variety of nontranslated RNA genes encoding transfer RNAs, 5S ribosomal RNA, U6 snRNA, and other small cellular RNAs (1). In Saccharomyces cerevisiae, transcription of these genes is directed by the initiation factor TFIIIB, which is assembled upstream of the start site by other factors (TFIIIA and͞or TFIIIC), bound to downstream promoter elements. Yeast TFIIIB is a heterotrimeric complex comprising the TATA-binding protein (TBP), a TFIIB-related component, TFIIIB70 (Brf1), and a SANT domain protein, TFIIIB90 (BЈЈ). Structural and functional homologs of these proteins have been identified in human cells and confer TFIIIB activity (termed TFIIIB-␣ in ref.2) for the transcription of tRNA and related pol III genes having internal promoter elements (2-4). Additionally, human cells contain a second TFIIIB activity (TFIIIB-␤) that is used by pol III genes whose promoter elements are located upstream of the start site (e.g., U6 snRNA and 7SK RNA). TFIIIB-␤ differs from TFIIIB-␣ in that it contains a different TFIIB-related component (termed BRFU or hTFIIIB50) and associated proteins (2, 3). Further complexity among TFIIIB complexes is suggested by the identification of three splice variants of human Brf1 (4). One of these variants, Brf2, appears to be active in the transcription U6 snRNA.Pol III genes are among the most actively transcribed genes in eukaryotic cells. High rates of pol III gene transcription are achieved through the facilitated recycling of pol molecules (5-8) on TFIIIB complexes that remain bound to the DNA for multiple rounds of initiation (9,10). The stability of TFIIIB-DNA complexes is therefore a key property that enables rapid reinitiation by eliminating rate-limiting steps in transcription complex assembly. Yeast TFIIIB-DNA compl...