isopropyl-thiogalactoside and 2-nitrophenyl-beta-fucoside

Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.

Topics: DNA-Directed RNA Polymerases; DNA, Bacterial; DNA, Superhelical; Escherichia coli; Gene Expression Regulation, Bacterial; Glucose; Glycosides; Isopropyl Thiogalactoside; Kinetics; Lac Operon; Plasmids; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Rifampin; RNA, Bacterial; Transcription, Genetic

Specific interactions between proteins and ligands that modify their functions are crucial in biology. Here, we examine sugars that bind the lactose repressor protein (LacI) and modify repressor affinity for operator DNA using isothermal titration calorimetry and equilibrium DNA binding experiments. High affinity binding of the commonly-used inducer isopropyl-beta,D-thiogalactoside is strongly driven by enthalpic forces, whereas inducer 2-phenylethyl-beta,D-galactoside has weaker affinity with low enthalpic contributions. Perturbing the dimer interface with either pH or oligomeric state shows that weak inducer binding is sensitive to changes in this distant region. Effects of the neutral compound o-nitrophenyl-beta,D-galactoside are sensitive to oligomerization, and at elevated pH this compound converts to an anti-inducer ligand with slightly enhanced enthalpic contributions to the binding energy. Anti-inducer o-nitrophenyl-beta,D-fucoside exhibits slightly enhanced affinity and increased enthalpic contributions at elevated pH. Collectively, these results both demonstrate the range of energetic consequences that occur with LacI binding to structurally-similar ligands and expand our insight into the link between effector binding and structural changes at the subunit interface.

Topics: Bacterial Proteins; Calorimetry; Dimerization; DNA; Glycosides; Hydrogen-Ion Concentration; Isopropyl Thiogalactoside; Lac Repressors; Ligands; Nitrophenylgalactosides; Operator Regions, Genetic; Protein Conformation; Repressor Proteins

The vast increase in available data from the "-omics" revolution has enabled the fields of structural proteomics and structure prediction to make great progress in assigning realistic three-dimensional structures to each protein molecule. The challenge now lies in determining the fine structural details that endow unique functions to sequences that assume a common fold. Similar problems are encountered in understanding how distinct conformations contribute to different phases of a single protein's dynamic function. However, efforts are hampered by the complexity of these large, three-dimensional molecules. To overcome this limitation, structural data have been recast as two-dimensional networks. This analysis greatly reduces visual complexity but retains information about individual residues. Such diagrams are very useful for comparing multiple structures, including (1) homologous proteins, (2) time points throughout a dynamics simulation, and (3) functionally different conformations of a given protein. Enhanced structural examination results in new functional hypotheses to test experimentally. Here, network representations were key to discerning a difference between unliganded and inducer-bound lactose repressor protein (LacI), which were previously presumed to be identical structures. Further, the interface of unliganded LacI was surprisingly similar to that of the K84L variant and various structures generated by molecular dynamics simulations. Apo-LacI appears to be poised to adopt the conformation of either the DNA- or inducer-bound structures, and the K84L mutation appears to freeze the structure partway through the conformational transition. Additional examination of the effector binding pocket results in specific hypotheses about how inducer, anti-inducer, and neutral sugars exert their effects on repressor function.

Topics: Bacterial Proteins; Combinatorial Chemistry Techniques; Crystallography, X-Ray; DNA-Binding Proteins; DNA, Bacterial; Glycosides; Isopropyl Thiogalactoside; Lac Operon; Lac Repressors; Operator Regions, Genetic; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Proteomics; Repressor Proteins; Structural Homology, Protein; Structure-Activity Relationship; Surface Properties; Thermodynamics

Crystal structures of the Lac repressor, with and without isopropyithiogalactoside (IPTG), and the repressor bound to operator have provided a model for how the binding of the inducer reduces the affinity of the repressor for the operator. However, because of the low resolution of the operator-bound structure (4.8 A), the model for the allosteric transition was presented in terms of structural elements rather than in terms of side chain interactions. Here we have constructed a dimeric Lac repressor and determined its structure at 2.6 A resolution in complex with a symmetric operator and the anti-inducer orthonitrophenylfucoside (ONPF). The structure enables the induced (IPTG-bound) and repressed (operator-bound) conformations of the repressor to be compared in atomic detail. An extensive network of interactions between the DNA-binding and core domains of the repressor suggests a possible mechanism for the allosteric transition.

Topics: Allosteric Regulation; Bacterial Proteins; Binding Sites; Crystallization; Crystallography, X-Ray; Dimerization; DNA; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Glycosides; Hydrogen Bonding; Isopropyl Thiogalactoside; Lac Repressors; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Operator Regions, Genetic; Protein Conformation; Repressor Proteins; Sequence Deletion; Static Electricity; Structure-Activity Relationship

The wild type E. coli lac operator is embedded in a 35 base-pair DNA sequence containing extensive 2-fold symmetry, suggesting a symmetric repressor operator complex. However, deviations from strict 2-fold symmetry occur at the central base-pair and at three additional base-pairs. Using an operator fragment binding analysis we have determined: (a) a relative contribution each pair provides to the lac repressor-lac operator DNA complex, (b) the operator DNA length necessary for maximum binding to lac repressor; and (c) the contribution of the several non-symmetric base in the wild-type operator to the binding affinity. Since lac repressor-lac operator DNA interaction is reduced upon binding of the gratuitous inducer, isopropyl-beta-D-galactoside (IPTG), the same DNA fragment binding analysis was performed with the low affinity form of lac repressor. In the presence of inducer, the affinity for the left half site of the wild-type lac operator is reduced without significant reduction on the right half of the operator. Conversely, the anti-inducer orthonitrophenylfucoside (ONPF) which stabilizes the lac repressor-lac operator complex increases the binding affinity, particularly to the right half of the operator.

Topics: Allosteric Regulation; Bacterial Proteins; Base Composition; Base Sequence; Binding Sites; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Glycosides; Isopropyl Thiogalactoside; Lac Operon; Lac Repressors; Molecular Sequence Data; Repressor Proteins; Thermodynamics

The thermal denaturation of the core protein of lac repressor was studied alone and in the presence of the inducer isopropyl beta-D-thiogalactoside (IPTG) and the antiinducer o-nitrophenyl beta-D-fucoside (ONPF) by means of high-sensitivity differential scanning calorimetry. The denaturation that takes place at about 65 degrees C is apparently irreversible; i.e., a rescan of a previously scanned sample of protein solution shows no denaturational endotherm. Despite this irreversibility, the denaturation appeared to follow quantitatively the dictates of equilibrium thermodynamics as embodied in the van't Hoff equation. The results obtained indicate clearly that the tetrameric protein dissociates to monomers during denaturation and that the ligands are not dissociated until denaturation takes place. The enthalpy of denaturation of the protein is 4.57 +/- 0.25 cal g-1 and is independent of temperature. The enthalpies of dissociation of IPTG and ONPF at the denaturation temperature are very large, 37 and 42 kcal (mol of ligand)-1, respectively.

Topics: Calorimetry, Differential Scanning; Escherichia coli; Glycosides; Isopropyl Thiogalactoside; Ligands; Protein Conformation; Protein Denaturation; Repressor Proteins; Thermodynamics; Transcription Factors