María-Trinidad Gallegos

The Bacterial Enhancer-Dependent ς54(ςN) Transcription Factor

The initiation of transcription is a complex process involving many different steps. These steps are all potential control points for regulating gene expression, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing

Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression.

The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 μm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (KD of around 1 μm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

Different Modes of Binding of Mono- and Biaromatic Effectors to the Transcriptional Regulator TTGV ROLE IN DIFFERENTIAL DEREPRESSION FROM ITS COGNATE OPERATOR

Members of the IclR family of regulators exhibit a highly conserved effector recognition domain and interact with a limited number of effectors. In contrast with most IclR family members, TtgV, the transcriptional repressor of the TtgGHI efflux pump, exhibits multidrug recognition properties. A three-dimensional model of the effector domain of TtgV was generated based on the available three-dimensional structure of several IclR members, and a series of point mutants was created. Using isothermal titration calorimetry, we determined the binding parameters of the most efficient effectors for TtgV and its mutant variants. All mutants bound biaromatic compounds with higher affinity than the wild-type protein, whereas monoaromatic compounds were bound with lower affinity. This tendency was particularly pronounced for mutants F134A and H200A. TtgVF134A bound 4-nitrotoluene with an affinity 13-fold lower than that of TtgV (17.4 ± 0.6 μm). This mutant bound 1-naphthol with an affinity of 5.7 μm, which is seven times as great as that of TtgV (40 μm). The TtgVV223A mutant bound to DNA with the same affinity as the wild-type TtgV protein, but it remained bound to the target operator in the presence of effectors, suggesting that Val-223 could be part of an intra-TtgV signal recognition pathway. Thermodynamic analyses of the binding of effectors to TtgV and to its mutants in complex with their target DNA revealed that the binding of biaromatic compounds resulted in a more efficient release of the repressor protein than the binding of monoaromatics. The physiological significance of these findings is discussed.

FUNCTIONS OF THE SIGMA 54 REGION I IN TRANS AND IMPLICATIONS FOR TRANSCRIPTION ACTIVATION

Control of transcription frequently involves the direct interaction of activators with RNA polymerase. In bacteria, the formation of stable open promoter complexes by the sigma(54) RNA polymerase is critically dependent on sigma(54) amino Region I sequences. Their presence correlates with activator dependence, and removal allows the holoenzyme to engage productively with melted DNA independently of the activator. Using purified Region I sequences and holoenzymes containing full-length or Region I-deleted sigma(54), we have explored the involvement of Region I in transcription activation. Results show that Region I in trans inhibits a reversible conformational change in the holoenzyme believed to be polymerase isomerization. Evidence is presented indicating that the holoenzyme (and not the promoter DNA per se) is one interacting target used by Region I in preventing polymerase isomerization. Activator overcomes this inhibition in a reaction requiring nucleotide hydrolysis. Region I in trans is able to inhibit activated transcription by the holoenzyme containing full-length sigma(54). Inhibition appeared to be noncompetitive with respect to the activator, suggesting that a direct activator interaction occurs with parts of the holoenzyme outside Region I. Stabilization of isomerized holoenzyme bound to melted DNA by Region I in trans occurs largely independently of the initiating nucleotide, suggesting a role for Region I in maintaining the open complex.

Functions of the ς54 Region I in Transand Implications for Transcription Activation

Control of transcription frequently involves the direct interaction of activators with RNA polymerase. In bacteria, the formation of stable open promoter complexes by the ς54 RNA polymerase is critically dependent on ς54 amino Region I sequences. Their presence correlates with activator dependence, and removal allows the holoenzyme to engage productively with melted DNA independently of the activator. Using purified Region I sequences and holoenzymes containing full-length or Region I-deleted ς54, we have explored the involvement of Region I in transcription activation. Results show that Region Iin trans inhibits a reversible conformational change in the holoenzyme believed to be polymerase isomerization. Evidence is presented indicating that the holoenzyme (and not the promoter DNAper se) is one interacting target used by Region I in preventing polymerase isomerization. Activator overcomes this inhibition in a reaction requiring nucleotide hydrolysis. Region Iin trans is able to inhibit activated transcription by the holoenzyme containing full-length ς54. Inhibition appeared to be noncompetitive with respect to the activator, suggesting that a direct activator interaction occurs with parts of the holoenzyme outside Region I. Stabilization of isomerized holoenzyme bound to melted DNA by Region I in trans occurs largely independently of the initiating nucleotide, suggesting a role for Region I in maintaining the open complex.

Involvement of the sigmaN DNA-binding domain in open complex formation.

σN (σ54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniaeσN mutants, altered in the carboxy DNA‐binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator‐dependent transcription. The mutant holoenzymes have altered activator‐dependent interactions with promoter sequences that normally become melted. Activator‐dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator‐independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I σN, suggesting different defects. The DNA‐binding domain substitutions are within σN sequences previously shown to be buried upon formation of the wild‐type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the σN–core and σN–DNA interfaces has occurred to change holoenzyme activity. Core‐binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of σN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator‐dependent transcription, formed a stable activator‐independent holoenzyme promoter complex on premelted DNA templates.

Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus.

The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor sigma(N) (sigma(54), RpoN) and five potential sigma(N)-dependent transcriptional activators. Although A. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative sigma(N)-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purified A. aeolicus sigma(N) and the NifA homologue, AQ_218. Purified A. aeolicus sigma(N) bound to E. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative sigma(N)-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus sigma(N) supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspFDeltaHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the sigma(N) enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and sigma(N) have coevolved.

Enzymatic Activation of the cis-trans Isomerase and Transcriptional Regulation of Efflux Pumps in Solvent Tolerance in Pseudomonas Putida

Organic solvents with a log Pow(logarithm of its partition coefficient in n-octanol and water) between 1.5 and 4.0 are extremely toxic for microorganisms and other living cells because they partition preferentially in the cytoplasmic membrane, disorganizing its structure and impairing vital functions99. The toxicity of these compounds depends not only on the inherent toxicity of the solvent but also on the intrinsic tolerance of the bacterial species and strains. The level of organic solvent tolerance in Escherichia coli is variable among strains; that is, E. coli JA300 and MC1061 grow in the presence of n-hexane (log Pow 3.9) whereas E. coli MVl184 or DH1 do not1, 2, and it has been proved to be enhanced by mutations. Most Pseudomonas species are highly sensit ive to aromatic hydrocarbons such as toluene (log Pow 2.5), styrene (log Pow 3.05) and p-xylene (log Pow3.2); however, independent laboratories have isolated Pseudomonas putida strains tolerant to these toxic compounds10, 37, 52, 80, 106.

Use of the tryptophan analogue 7-azatryptophan to study the interaction of σN with Escherichia coli RNA polymerase core enzyme

The minor sigma factor σ N of Escherichia coli RNA polymerase (RNAP) has been labelled with the tryptophan analogue 7-azatryptophan (7AW) by biosynthetic incorporation. This has the effect of shifting the absorbance spectra of the protein so there is appreciable absorbance at 315 nm. Consequently this makes 7AWσ N an ideal tool to study the protein/protein interaction of σ N and RNAP using the absorbance optics in the XL-A analytical ultracentrifuge.