The synthesis of potential antimalarials. Derivatives of pantoyltaurine

The general hypothesis as to the mode of action of chemotherapeutic agents, which has been formulated by Fildes, Woods, McIlwain, and others (2), offers a rational and useful guide to the design of new drugs. Thus, bacteriostasis is pictured as caused by the blocking of reactions essential to growth by an inhibiting substance which has a structure similar to that of one of the normal enzymes or metabolites essential to the growth of the organism

Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy

Rapid binding and release of Hfq from ternary complexes during RNA annealing

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼108 M−1s−1) observed with Cy3-Hfq was followed by a slow transition (0.5 s−1) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions

The Cooperative Binding Energetics of CytR and cAMP Receptor Protein Support a Quantitative Model of Differential Activation and Repression of CytR-Regulated Class III <i>Escherichia coli</i> Promoters

cAMP receptor protein (CRP) and CytR mediate positive and negative control of nine genes in <i>Escherichia coli</i>, most of which are involved in nucleoside catabolism and recycling. Five promoters share a common architecture in which tandem CRP sites flank an intervening CytR operator (CytO). CytR and CRP bind cooperatively to these promoters to form a three-protein, DNA-bound complex that controls activation and repression, the levels of which vary markedly among the promoters. To understand the specific combinatorial control mechanisms that are responsible for this outcome, we have used quantitative DNase I footprinting to generate individual site isotherms for each site of protein–DNA interaction. The intrinsic affinities of each transcription factor for its respective site and the specific patterns of cooperativity and competition underlying the molecular interactions at each promoter were determined by a global analysis of these titration data. Here we present results obtained for <i>nupGP</i> and <i>tsxP2</i>, adding to results published previously for <i>deoP2</i>, <i>udpP</i>, and <i>cddP</i>. These data allowed us to correlate the reported levels of activation, repression, and induction with the ligation states of these five promoters under physiologically relevant conditions. A general pattern of transcriptional regulation emerges that allows for complex patterns of regulation in this seemingly simple system