Donald F. Senear

[9] Quantitative DNase footprint titration: A method for studying protein-DNA interactions

Publisher Summary This chapter discusses that individual-site binding isotherms are uniquely suited to permit the resolution of interaction parameters for systems exhibiting cooperative interactions between multiple sites. The analysis is completely general. Any number of specific sites can be analyzed regardless of the nature of the cooperative or anticooperative interactions among them. The development of the footprint titration method, which permits resolution of individual-site isotherms, permits quantitative characterization of systems that act as critical regulators of gene transcription. The thermodynamic parameters that are resolved from footprint titration can be used in two important ways to further the understanding of gene regulation. First, the binding affinities of the various components of a gene regulatory system can be used to deduce the mechanism of the regulation—for example, the successful modeling of the switch from the lysogenic-to-lytic growth stage of the lambda phage. Second, the range of precisely controlled experimental conditions over which the technique is applicable allows to measure other thermodynamic parameters—for example, enthalpies and entropies—to study the roles of the various noncovalent forces of interaction involved in protein-DNA binding and site recognition.

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.

DNase I Footprint Analysis of Protein‐DNA Binding

Deoxyribonuclease I (DNase I) protection mapping, or footprinting, is a valuable technique for locating the specific binding sites of proteins on DNA. The basis of this assay is that bound protein protects the phosphodiester backbone of DNA from DNase I‐catalyzed hydrolysis. Binding sites are visualized by autoradiography of the DNA fragments that result from hydrolysis, following separation by electrophoresis on denaturing DNA sequencing gels. Footprinting has been developed further as a quantitative technique to determine separate binding curves for each individual protein‐binding site on the DNA. For each binding site, the total energy of binding is determined directly from that sites binding curve. For sites that interact cooperatively, simultaneous numerical analysis of all the binding curves can be used to resolve both the intrinsic binding and cooperative components of these energies.

Activation of Gene Expression by a Ligand-induced Conformational Change of a Protein-DNA Complex

IlvY protein binds cooperatively to tandem operator sites in the divergent, overlapping, promoter-regulatory region of the ilvYC operon of Escherichia coli. IlvY positively regulates the expression of the ilvC gene in an inducer-dependent manner and negatively regulates the transcription of its own divergently transcribed structural gene in an inducer-independent manner. Although binding of IlvY protein to the tandem operators is sufficient to repress ilvYpromoter-specific transcription, it is not sufficient to activate transcription from the ilvC promoter. Activation ofilvC promoter-specific transcription requires the additional binding of a small molecule inducer to the IlvY protein-DNA complex. The binding of inducer to IlvY protein does not affect the affinity of IlvY protein for the tandem operator sites. It does, however, cause a conformational change of the IlvY protein-DNA complex, which is correlated with the partial relief of an IlvY protein-induced bend of the DNA helix in the ilvC promoter region. This structural change in the IlvY protein-DNA complex results in a 100-fold increase in the affinity of RNA polymerase binding at theilvC promoter site. The ability of a protein to regulate gene expression by ligand-responsive modulation of a protein-DNA structure is an emerging theme in gene regulation.

Simultaneous analysis for testing of models and parameter estimation

Publisher Summary This chapter describes the application of the least-squares method by providing two examples of current problems, which benefit from simultaneous analyses. The two examples are the linear extrapolation method for determination of protein unfolding free energy changes and the individual site binding approach for the determination of protein–DNA interaction free energy changes. In the former case, it is found that by properly formulating the model to apply the least-squares criterion to baseline extensions and extrapolation of free energy changes, more realistic limits to the estimate of the unfolding free energy change are obtained. Simultaneous analysis of the effects of different denaturants increases the precision of the estimates and provides confidence in some of the assumptions of the model. In the later case, the proper combination of data for binding of a repressor ligand to a multisite operator DNA and to reduced valency mutants is necessary to resolve all of the interaction free energy changes. The chapter introduces nonlinear least-squares techniques, which provide direct means of analysis for complex models and illustrates by example the powerful technique of simultaneous analysis for resolving parameters and testing the adequacy of the model used by the investigator.

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.

Allosteric Mechanism of Induction of CytR-regulated Gene Expression CytR REPRESSOR-CYTIDINE INTERACTION

Transcription from cistrons of theEscherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR. De-repression results when CytR binds cytidine. CytR is a homodimer and a LacI family member. A central question for all LacI family proteins concerns the allosteric mechanism that couples ligand binding to the protein-DNA and protein-protein interactions that regulate transcription. To explore this mechanism for CytR, we analyzed nucleoside binding in vitro and its coupling to cooperative CytR binding to operator DNA. Analysis of the thermodynamic linkage between sequential cytidine binding to dimeric CytR and cooperative binding of CytR to deoP2 indicates that de-repression results from just one of the two cytidine binding steps. To test this conclusion in vivo, CytR mutants that have wild-type repressor function but are cytidine induction-deficient (CID) were identified. Each has a substitution for Asp281or neighboring residue. CID CytR281N was found to bind cytidine with three orders of magnitude lower affinity than wild-type CytR. Other CytR mutants that do not exhibit the CID phenotype were found to bind cytidine with affinity similar to wild-type CytR. The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions. The data support the conclusion that the first cytidine binding step alone is sufficient to induce.

Multiple Specific CytR Binding Sites at the Escherichia coli deoP2 Promoter Mediate Both Cooperative and Competitive Interactions between CytR and cAMP Receptor Protein

Binding of cAMP receptor protein (CRP) and CytR mediates both positive and negative control of transcription from Escherichia coli deoP2. Transcription is activated by CRP and repressed by a multi-protein CRP·CytR·CRP complex. The latter is stabilized by cooperative interactions between CRP and CytR. Similar interactions at the other transcriptional units of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. To understand the combinatorial control mechanism at deoP2, we have used quantitative footprint and gel shift analysis of CRP and CytR binding to evaluate the distribution of ligation states. By comparison to distributions for other CytR-regulated promoters, we hope to understand the roles of individual states in differential gene expression. The results indicate that CytR binds specifically to multiple sites at deoP2, including both the well recognized CytR site flanked by CRP1 and CRP2 and also sites coincident with CRP1 and CRP2. Binding to these multiple sites yields both cooperative and competitive interactions between CytR and CRP. Based on these findings we propose that CytR functions as a differential modulator of CRP1 versus CRP2-mediated activation. Additional high affinity specific sites are located at deoP1 and near the middle of the 600-base pair sequence separating P1 and P2. Evaluation of the DNA sequence requirement for specific CytR binding suggests that a limited array of contiguous and overlapping CytR sites exists at deoP2. Similar extended arrays, but with different arrangements of overlapping CytR and CRP sites, are found at the other CytR-regulated promoters. We propose that competition and cooperativity in CytR and CRP binding are important to differential regulation of these promoters.

Role of multiple CytR binding sites on cooperativity, competition, and induction at the Escherichia coli udp promoter.

The CytR repressor fulfills dual roles as both a repressor of transcription from promoters of the Escherichia coli CytR regulon and a co-activator in some circumstances. Transcription is repressed by a three-protein complex (cAMP receptor protein (CRP)-CytR-CRP) that is stabilized by cooperative interactions between CRP and CytR. However, cooperativity also means that CytR can recruit CRP and, by doing so, can act as a co-activator. The central role of cooperativity in regulation is highlighted by the fact that binding of the inducer, cytidine, to CytR is coupled to CytR-CRP cooperativity; this underlies the mechanism for induction. Similar interactions at the different promoters of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism but also provide differential expression of these genes. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. Recently, we showed that CytR binds specifically to multiple sites in the E. coli deoPpromoter, thereby providing competition for CRP binding to CRP operator site 1 (CRP1) and CRP2 as well as cooperativity. The effect of the competition at this promoter is to negate the role of CytR in recruiting CRP. Here, we have used quantitative footprint and mobility shift analysis to investigate CRP and CytR binding to theE. coli udp promoter. Here too, we find that CytR both cooperates and competes for CRP binding. However, consistent with both the distribution of CytR recognition motifs in the sequence of the promoter and the regulation of the promoter, the competition is limited to CRP2. When cytidine binds to CytR, the effect on cooperativity is very different at the udp promoter than at thedeoP2 promoter. Cooperativity with CRP at CRP1 is nearly eliminated, but the effect on CytR-CRP2 cooperativity is negligible. These results are discussed in relation to the current structural model of CytR in which the core, inducer-binding domain is tethered to the helix-turn-helix, DNA-binding domain via flexible peptide linkers.

Quantitative DNase I Footprinting

Publisher Summary This chapter provides an overview on quantitative deoxyribonuclease I (DNase I) footprinting. DNase I footprinting was introduced by Schmitz and Galas to identify the DNA sequences that constitute binding sites for site-specific DNA-binding proteins. It has been used to detect changes in DNA structure and to measure the relative binding affinities of DNA-binding proteins. The unique value of footprinting as a quantitative titration technique for protein-DNA interactions is its ability to separately monitor the binding of protein to each specific site on the DNA. Further, the chapter focuses on the use of DNase I footprinting in vitro to yield data about specific binding sites and to obtain thermodynamic information that characterizes DNA-protein systems. One advantage of DNase I over chemical footprinting agents is that its enzymatic activity is specific to the DNA, so that degradation of the protein ligand is not a concern. A disadvantage is that its activity is somewhat sensitive to the DNA sequence, so that some regions are inefficiently cleaved even in the absence of bound protein ligand.