Biomedical subjects

Donald F Senear

Publications and source records attributed to Donald F Senear.

6 recordsLinked to original sources

PubMed2006

Indirect recognition in sequence-specific DNA binding by Escherichia coli integration host factor: the role of DNA deformation energy.

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.

Amino Acid Motifs↗

PubMed2006

Flexibility and adaptability in binding of E. coli cytidine repressor to different operators suggests a role in differential gene regulation.

Interactions between DNA-bound transcription factors CytR and CRP regulate the promoters of the Escherichia coli CytR regulon. A distinctive feature of the palindromic CytR operators is highly variable length central spacers (0-9 bp). Previously we demonstrated distinct modes of CytR binding to operators that differ in spacer length. These different modes are characterized by opposite enthalpic and entropic contributions at 25 degrees C. Of particular note were radically different negative DeltaCp values suggesting variable contribution from coupled protein folding and/or DNA structural transitions. We proposed that the CytR DNA binding-domain adopts either a more rigid or flexible DNA-bound conformation in response to the different spacer lengths. More recently, similar effects were shown to contribute to discrimination between operator and non-specific DNA binding by LacR, a CytR homolog. Here we have extended the thermodynamic analysis to the remaining natural CytR operators plus a set of synthetic operators designed to isolate spacing as the single variable. The thermodynamic results show a broad and monotonic range of effects that are primarily dependent on spacer length. The magnitude of effects suggests participation by more than the DNA-binding domain. 15N HSQC NMR and CD spectral analyses were employed to characterize the structural basis for these effects. The results indicate that while CytR forms a well-ordered structure in solution, it is highly dynamic. We propose a model in which a large ensemble of native state conformations narrows upon binding, to an extent governed by operator spacing. This in turn is expected to constrain intermolecular interactions in the CytR-CRP-DNA complex, thus generating operator-specific effects on repression and induction of transcription.

Cytidine↗

PubMed2003

Role of protein-protein bridging interactions on cooperative assembly of DNA-bound CRP-CytR-CRP complex and regulation of the Escherichia coli CytR regulon.

The unlinked operons that comprise the Escherichia coli CytR regulon are controlled coordinately through interactions between two gene regulatory proteins, the cAMP receptor protein (CRP) and the cytidine repressor (CytR). CytR controls the balance between CRP-mediated recruitment and activation of RNA polymerase and transcriptional repression. Cooperative interactions between CytR, when bound to an operator (CytO) located upstream of a CytR-regulated promoter, and CRP, when bound to flanking tandem promoters, are critical to the regulatory role of CytR. When CytR binds cytidine, cooperativity is reduced resulting in increased transcriptional activity. However, this cytidine-mediated effect varies among promoters, suggesting that coupling between cytidine binding to CytR and CytR-CRP association is sensitive to promoter structure. To investigate the chemical and structural basis for these effects, we investigated how cytidine binding affects association between CytR and CRP in solution and how it affects the binding of CytR deletion mutants lacking the DNA binding HTH domain, with tandem CRP dimers bound to either udpP or deoP2. Deletion mutants that, as we show here, retain the native functions of the allosteric, inducer-binding domain but do not bind DNA were expressed and purified. We refer to these as Core domain. Despite only weak association between CytR and CRP in solution, our results demonstrate the formation of a relatively stable complex in which the Core domain forms a protein bridge between tandem CRP dimers when bound to either udpP or deoP2. The DeltaG(o) for bridge complex formation is about -7.8 kcal/mol. This is well in excess of that required to account for cooperativity (-2.5 to -3 kcal/mol). The bridge complexes are significantly destabilized by cytidine binding, and to the same extent in both promoter complexes (DeltaDeltaG(o) approximately +2 kcal/mol). Even with this destabilization, DeltaG(o) for bridge complex formation by cytidine-liganded Core domain is still sufficient by itself to account for cooperativity. These findings demonstrate that direct coupling between cytidine binding to CytR and CytR-CRP association does not account for promoter-specific effects on cooperativity. Instead, cytidine binding must induce a CytR conformation that is more rigid or in some other way less tolerant of the variation in the geometric arrangement of operator sites between different promoters.

Binding Sites↗

PubMed2002

Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.

The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated.

Binding Sites↗

PubMed2002

Thermodynamics of E. coli cytidine repressor interactions with DNA: distinct modes of binding to different operators suggests a role in differential gene regulation.

Interactions between the Escherichia coli cytidine repressor protein (CytR) and its operator sites at the different promoters that comprise the CytR regulon, play an important role in the regulation of these promoters. The natural operators are palindromes separated by variable length central spacers (0-9 bp). We have suggested that this variability affects the flexibility of CytR-DNA contacts, thereby affecting the critical protein-protein interactions between CytR and the cAMP receptor protein (CRP) that underlie differential repression and activation of CytR-regulated genes. To assess this hypothesis, we investigated the thermodynamics of CytR binding to the natural operator sequences found in udpP and deoP2. To separate effects due to spacing from effects due to the differing sequences of the recognition half-sites of these two operators, we also investigated CytR binding to artificial hybrid operators, in which the half-site sequences of udpP and deoP2 were exchanged. Thermodynamic parameters, DeltaS(o), DeltaH(o) and DeltaC(o)(p), were determined by van't Hoff analysis of CytR binding, monitored by changes in the steady-state fluorescence anisotropy of dye-conjugated, operator-containing oligonucleotides. Large differences in thermodynamics were observed that depend primarily on the central spacer rather than the sequences of the recognition half-sites. Binding to operators with deoP2 spacing results in a very large, negative DeltaC(o)(p). Association is strongly favored enthalpically and strongly disfavored entropically at ambient temperature. By contrast, binding to operators with udpP spacing results in a small, negative DeltaC(o)(p). Association is weakly favored both enthalpically and entropically at ambient temperature. A difference of such magnitude in DeltaDeltaC(o)(p) has not been reported previously for specific binding of a transcription factor to different sites. The identical salt dependence of CytR binding to deoP2 and udpP operators indicates that ion-dependent processes do not contribute significantly to this difference. Thus, the different thermodynamic effects appear to reflect distinctly different modes of site-specific DNA binding. We discuss similarities to operator binding by CytR homologs among LacI family repressors, and we consider how different CytR binding modes might affect interactions with other components of the gene regulatory machinery that contribute to differential gene regulation.

Base Sequence↗

PubMed2002

Quantitative analysis of tryptophan analogue incorporation in recombinant proteins.

Three different methods to quantitate tryptophan (Trp) analogue incorporation into recombinant proteins are described: first, spectroscopic analysis based on a linear combination of the absorption spectra of the aromatic residues in the denatured Trp-containing or analogue-substituted protein; second, chromatographic separation of analogue-substituted and Trp-containing proteins by HPLC; and third, mass spectrum analysis of the mixture of analogue-substituted and Trp-containing proteins. An accurate estimate of analogue incorporation in single-Trp proteins can be obtained directly by either analysis of the absorption spectrum or HPLC chromatography. While analysis of the absorption spectrum or HPLC chromatogram can provide an assessment of the average level of analogue incorporation for proteins that contain two or more Trp residues, mass spectroscopy analysis of peptides generated by protease digestion and separated by HPLC provides a general method for a complete quantitative description of the distribution of analogue incorporation. The more complex analysis by mass spectroscopy becomes important for multi-Trp proteins because the distribution of analogue versus Trp-containing polypeptide chains may not be the same as that predicted on the basis of average level of analogue incorporation.

Chromatography, High Pressure Liquid↗