Modeling helix-turn-helix protein-induced DNA bending with knowledge-based distance restraints.

A crucial element of many gene functions is protein-induced DNA bending. Computer-generated models of such bending have generally been derived by using a presumed bending angle for DNA. Here we describe a knowledge-based docking strategy for modeling the structure of bent DNA recognized by a major groove-inserting alpha-helix of proteins with a helix-turn-helix (HTH) motif. The method encompasses a series of molecular mechanics and dynamics simulations and incorporates two experimentally derived distance restraints: one between the recognition helix and DNA, the other between respective sites of protein and DNA involved in chemical modification-enabled nuclease scissions. During simulation, a DNA initially placed at a distance was "steered" by these restraints to dock with the binding protein and bends. Three prototype systems of dimerized HTH DNA binding were examined: the catabolite gene activator protein (CAP), the phage 434 repressor (Rep), and the factor for inversion stimulation (Fis). For CAP-DNA and Rep-DNA, the root mean square differences between model and x-ray structures in nonhydrogen atoms of the DNA core domain were 2.5 A and 1.6 A, respectively. An experimental structure of Fis-DNA is not yet available, but the predicted asymmetrical bending and the bending angle agree with results from a recent biochemical analysis.     

A divalent metal-mediated switch controlling protein-induced DNA bending

Architectural proteins that reconfigure the paths of DNA segments are required for the establishment of functional interfaces in many genomic transactions. A single-chain derivative of the DNA architectural protein integration host factor was found to adopt two stable conformational states in complex with a specific DNA target. In the so-called open state, the degree of protein-induced DNA bending is reduced significantly compared with the closed state. The conformational switch between these states is controlled by divalent metal binding in two electronegative zones arising from the lysine-to-glutamate substitution in the protein body proximal to the phosphate backbone of one DNA arm. We show that this switch can be employed to control the efficiency of site-specific recombination catalyzed by lambda integrase. Introduction of acidic residues at the protein-DNA interface holds potential for the design of metal-mediated switches for the investigation of functional relationships.     

Replacement of integration host factor protein-induced DNA bending by flexible regions of DNA

The Escherichia coli integration host factor (IHF) protein is required for site-specific recombination of bacteriophage lambda DNA. Previously, we had shown that alternative modules of static DNA curvature could partially replace IHF in recombination. Now we use regions of single-stranded DNA as a flexible tether to address whether the function of IHF in recombination is simply to reduce persistence length. Although we find that these modules clearly enhance recombination in the absence of IHF, they are not perfect replacements. In addition, evidence is presented that the efficacy of a flexibility swap is specific to a particular IHF site. This may indicate that additional functions beyond simple deformation of DNA are required of IHF. During the course of these experiments we discovered that these flexible sequences are still specific sites for IHF binding and function.     

(+)-CC-1065 as a probe for intrinsic and protein-induced bending of DNA

(+)-CC -1065 is biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. This antibiotic covalently modifies DNA by alkylation of N-3 of a adenine in the minor groove. As a Structural consequence of covalent modification of DNA, the helix axis id bent into the minor groove. The drug-induced bending of DNA has similarities to intrinsic. A-tract bending and the 3′ adenine of A-tracts shows a unique reactivity to alkylation by (+) -CC-1065. Upon covalent modification of A-tracts, the magnitude of bending is increased and helix is stiffened. Using high-field NMR, hydroxyl-radical footprinting and gel electrophoresis, the molecular basis for the high reactivity of the bonding sequence 5′ - AGTTA* (an asterisk indicates the covalent modification site) to (+)-CC-1065 has been shown to involve the inherent conformational flexibility of this sequence. Furthermore, these studies also demonstrate that after alkylation the drug-induced bending is focused over the TT region. By analogy with the junction bend model for A-tracts, a ‘truncated junction bend model’ is proposed for this structure. Last, the application of (+)-CC-1065 entrapped/induced bending of DNA as a probe for the Sp1-induced bending of the 21-base-pair repeat an Mu transpose bending of the att L3 sequence is described.     

RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex

Using high-resolution atomic force microscopy (AFM) we show that in a ternary complex of an activator protein, FIS, and RNA polymerase containing the sigma(70) specificity factor at the Escherichia coli tyrT promoter the polymerase and the activator form discrete, but connected, subcomplexes in close proximity. This is the first time that a ternary complex between an activator, a sigma(70) polymerase holoenzyme and promoter DNA has been visualised. Individually FIS and RNA polymerase wrap approximately 80 and 150 bp of promoter DNA, respectively. We suggest that the architecture of the ternary complex provides a general paradigm for the facilitation of direct, but weak, interactions between polymerase and an activator.