Thermodynamics of the interactions of Lac repressor with variants of the symmetric Lac operator: effects of converting a consensus site to a non-specific site

de, Frank; Rm, Saecker; Jp, Bond; Mw, Capp; Ov, Tsodikov; Se, Melcher; Mm, Levandoski; Mt, Record

doi:10.1006/jmbi.1997.0920

Cited by 112 publications

(99 citation statements)

References 105 publications

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“…2A left panel) (34)(35)(36). To surmount this limitation, we created the SSL as an adaptable tool to display and interpret the full recognition preferences of DNA binding molecules (Fig.…”

Section: Resultsmentioning

confidence: 99%

Specificity landscapes of DNA binding molecules elucidate biological function

Carlson

Warren

Hauschild

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

151

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Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes.Here we compare the DNA sequence specificities for different classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized and interpreted using an interactive "specificity landscape" which simultaneously displays the affinity and specificity of a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater accuracy in annotating regulatory elements within a given genome. Assigning such context-dependent binding values to every DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity of DNA binding molecules.

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“…2A left panel) (34)(35)(36). To surmount this limitation, we created the SSL as an adaptable tool to display and interpret the full recognition preferences of DNA binding molecules (Fig.…”

Section: Resultsmentioning

confidence: 99%

Specificity landscapes of DNA binding molecules elucidate biological function

Carlson

Warren

Hauschild

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

151

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“…For example, the presence of specific motif leads statistically, on average to ∼ − 2 k B T free-energy difference compared with nonspecific sequence (lacking such motif) for the Max protein used in our study. This free-energy difference reaches ∼ − 12 k B T for one of the most specific prokaryotic proteins, the lac repressor, LacI (32). The magnitude of the nonconsensus effect is expected to be significantly smaller in the latter case.…”

Section: Discussionmentioning

confidence: 97%

Protein−DNA binding in the absence of specific base-pair recognition

Afek

Schipper

Horton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

134

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Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)−DNA binding. Contrary to this assumption, here we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF−DNA binding preferences. We used highthroughput protein−DNA binding assays to measure the binding levels and free energies of binding for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein−DNA binding mechanism induced by DNA sequence symmetry in the absence of specific base-pair recognition, and experimentally demonstrate that this mechanism indeed governs protein−DNA binding preferences. protein−DNA binding is an important biophysical mechanism operating in a living cell (1). This seminal work makes it possible to interpret experiments that measured how transcription factors (TFs) search for their specific target sites flanked by nonconsensus sequence elements (1-10). A specific consensus motif is a short DNA sequence, typically 6-20 base pairs (bp), that possesses an enhanced binding affinity for a particular TF. For example, the sequence CACGTG represents the specific consensus motif for the human protein Max used in this study (Fig. 1). The process of establishing specific, consensus protein−DNA binding requires the formation of precise geometrical fit between the protein and its consensus DNA motif, accompanied by the formation of specific hydrogen and electrostatic contacts at the protein−DNA binding interface (6, 7) ( Fig. 1). In addition to binding to their consensus DNA motifs, transcription factors can also bind, albeit with lower affinity, to DNA regions lacking any consensus motifs. The term "nonspecific protein−DNA binding" (6) is typically used to describe these weaker interactions. Von Hippel and Berg suggested classifying nonspecific protein−DNA binding into two related mechanisms (6). The first mechanism includes protein binding to its mutated specific motifs that retain some residual, reduced specificity. The second mechanism is largely DNA sequence independent, and it involves electrostatic binding modulated by the overall DNA geometry (6). Despite significant experimental progress, molecular mechanisms responsible for these two types of nonspecific binding remain poorly understood, and the free energy of nonspecific protein−DNA binding has not been systematically characterized (11)(12)(13)(14). The interplay between consensus and nonconsensus DNA sequence elements emerges as a dominant factor that governs protein−DNA binding preferences. However, this interplay is also poorly understood (15, 16). Until now, it has been reasonably assumed that specific (consensus) base-pair recognition must control the genome-wide specificity of TF−DNA binding.Contrary to this assumption, here we identify a general mechanism for protein−DNA bi...

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“…Our primary interest is in the DNA binding specificity of the lac repressor. Measurements of affinity changes due to operator sequence variation, by base replacement, by the use of base analogs, or by changing the length of the operator, have been performed almost since the operator sequence was first determined (Goeddel et al 1978;Sadler et al 1983;Betz et al 1986;Sartorius et al 1989;Lehming et al 1990;Sasmor and Betz 1990;Frank et al 1997;Spronk et al 1999;Falcon and Matthews 2001;Kalodimos et al 2002Kalodimos et al , 2004bDaber and Lewis 2009). But those analyses all measured binding affinity to only a few sequences.…”

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confidence: 99%

“…As data accumulated showing that variations at different positions had different effects on binding, PWM models were developed to accommodate position-specific costs (reviewed in Stormo 2000; Stormo 2013), but those models still assume additivity. Quantitative analyses subsequently found that, while additivity is often violated (e.g., Stormo et al 1986;Frank et al 1997;Man and Stormo 2001;Bulyk et al 2002;Maerkl and Quake 2007;Zhao et al 2012;Weirauch et al 2013), it is often a surprisingly good approximation Takeda et al 1989;Benos et al 2002;Zhao et al 2012;Weirauch et al 2013), indicating that the average effect of each variant, which is captured in the PWM, is similar in most contexts.Deviations from additivity are often rather modest, ,0.5 kT between observed and predicted binding energies, so the data from high-throughput experiments may not be sufficiently accurate to distinguish between models. Spec-seq provides quite accurate relative binding energies, usually within $0.1 kT, allowing us to make reliable determinations of nonadditive contributions.…”

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confidence: 99%

High-Resolution Specificity from DNA Sequencing Highlights Alternative Modes of Lac Repressor Binding

Zuo¹,

Stormo

2014

Genetics

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Knowing the specificity of transcription factors is critical to understanding regulatory networks in cells. The lac repressoroperator system has been studied for many years, but not with high-throughput methods capable of determining specificity comprehensively. Details of its binding interaction and its selection of an asymmetric binding site have been controversial. We employed a new method to accurately determine relative binding affinities to thousands of sequences simultaneously, requiring only sequencing of bound and unbound fractions. An analysis of 2560 different DNA sequence variants, including both base changes and variations in operator length, provides a detailed view of lac repressor sequence specificity. We find that the protein can bind with nearly equal affinities to operators of three different lengths, but the sequence preference changes depending on the length, demonstrating alternative modes of interaction between the protein and DNA. The wild-type operator has an odd length, causing the two monomers to bind in alternative modes, making the asymmetric operator the preferred binding site. We tested two other members of the LacI/ GalR protein family and find that neither can bind with high affinity to sites with alternative lengths or shows evidence of alternative binding modes. A further comparison with known and predicted motifs suggests that the lac repressor may be unique in this ability and that this may contribute to its selection.T HE lactose regulatory system established the paradigm of a trans-acting factor binding to a cis-acting element to regulate the expression of the adjacent gene in response to an environmental signal (Jacob and Monod 1961). Many aspects of the lac repressor protein have been studied extensively (reviewed in Lewis 2005). Our primary interest is in the DNA binding specificity of the lac repressor. Measurements of affinity changes due to operator sequence variation, by base replacement, by the use of base analogs, or by changing the length of the operator, have been performed almost since the operator sequence was first determined (Goeddel et al. 1978;Sadler et al. 1983;Betz et al. 1986;Sartorius et al. 1989;Lehming et al. 1990;Sasmor and Betz 1990;Frank et al. 1997;Spronk et al. 1999;Falcon and Matthews 2001;Kalodimos et al. 2002Kalodimos et al. , 2004bDaber and Lewis 2009). But those analyses all measured binding affinity to only a few sequences.The lac repressor has not, to our knowledge, been analyzed by current high-throughput methods that can determine specificity over thousands, or even millions, of sequences in parallel (Stormo and Zhao 2010), such as protein-binding microarrays (PBM) (Berger et al. 2006;Gordan et al. 2013), SELEX-seq [or HT-SELEX (Zhao et al. 2009;Zykovich et al. 2009;Jolma et al. 2010;Wong et al. 2011)], bacterial one-hybrid (B1H) (Meng et al. 2005;Noyes et al. 2008;Christensen et al. 2011), and mechanically induced trapping of molecular interactions (MITOMI) (Maerkl and Quake 2007). While those methods offer an expansive overvie...

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Thermodynamics of the interactions of Lac repressor with variants of the symmetric Lac operator: effects of converting a consensus site to a non-specific site

Cited by 112 publications

References 105 publications

Specificity landscapes of DNA binding molecules elucidate biological function

Specificity landscapes of DNA binding molecules elucidate biological function

Protein−DNA binding in the absence of specific base-pair recognition

High-Resolution Specificity from DNA Sequencing Highlights Alternative Modes of Lac Repressor Binding

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