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...

We describe a new method for measuring the effects of epigenetic marks on protein-DNA interactions.

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B 12 biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.Global energy demands will increase significantly in coming decades (16), and renewable energy sources such as biofuels have been proposed to help reduce dependence upon fossil energy (8,23,27,49). Current efforts focus on biofuel production from renewable lignocellulosic feedstock (e.g., switchgrass), which constitutes ϳ50% of the world's biomass (2,8,23,27,49). Although intensive research and development have been performed on the effective utilization of lignocellulose, problems associated with practical use of this material have not been resolved fully (13). When enzymatic hydrolysis is adopted for cellulosic ethanol production, different levels of process integration can be visualized: (i) separate (or sequential) hydrolysis and fermentation (SHF), where the enzymes (cellulases) are used separately from fermentation tanks; (ii) simultaneous saccharification and fermentation (SSF), which consolidates enzymatic hydrolysis with fermentation of hexose or pentose; (iii) simultaneous saccharification and cofermentation (SSCF), which further combines the fermentation of hexose and pentose together; and (iv) consolidated bioprocessing (CBP), where all required enzymes and ethanol are produced in a single reactor (1,13,24,29,46,51). While these production schemes represent increasing levels of simplification through process consolidation, consolidation of multiple steps often results in a loss of process efficiency. Thus, improving the efficiency of individual steps, such as cellulose hydrolysis and ethanol fermentation, remains an important task for the development of economically feasible cellulosic bioethanol.Recent efforts have focused on metabolic engineering and (more recently) synthetic biology to produce strains or consortia capable of producing biofuels. Efforts to ...

The specificity of protein-DNA interactions can be determined directly by sequencing the bound and unbound fractions in a standard binding reaction. The procedure is easy and inexpensive, and the accuracy can be high for thousands of sequences assayed in parallel. From the measurements, simple models of specificity, such as position weight matrices, can be assessed for their accuracy and more complex models developed if useful. Those may provide more accurate predictions of in vivo binding sites and can help us to understand the details of recognition. As an example, we demonstrate new information gained about the binding of lac repressor. One can apply the same method to combinations of factors that bind simultaneously to a single DNA and determine both the specificity of the individual factors and the cooperativity between them.

Background: Feed-forward loops are utilized in glucocorticoid signaling and can bestow temporal control to gene regulation. Results: Cooperation with KLF15 enhances low affinity glucocorticoid receptor binding site activity in coherent feed-forward loops controlling amino acid catabolism. Conclusion: Feed-forward response element composition contributes to temporal diversity of transcriptional regulation by glucocorticoids. Significance: Cooperative feed-forward regulatory control may underpin glucocorticoid-induced metabolic side effects.

We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis—GRaMS (Growth Rate Modeling of Specificity)—that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well-characterized C2H2 zinc-finger TF on both a 28 bp randomized library for the standard B1H method and on 6 bp randomized library for the CV-B1H method for which 45 different experimental conditions were tested: five time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media.

Cooperative binding of transcription factors is known to be important in the regulation of gene expression programs conferring cellular identities. However, current methods to measure cooperativity parameters have been laborious and therefore limited to studying only a few sequence variants at a time. We developed Coop-seq (cooperativity by sequencing) that is capable of efficiently and accurately determining the cooperativity parameters for hundreds of different DNA sequences in a single experiment. We apply Coop-seq to 12 dimer pairs from the Sox and POU families of transcription factors using 324 unique sequences with changed half-site orientation, altered spacing and discrete randomization within the binding elements. The study reveals specific dimerization profiles of different Sox factors with Oct4. By contrast, Oct4 and the three neural class III POU factors Brn2, Brn4 and Oct6 assemble with Sox2 in a surprisingly indistinguishable manner. Two novel half-site configurations can support functional Sox/Oct dimerization in addition to known composite motifs. Moreover, Coop-seq uncovers a nucleotide switch within the POU half-site when spacing is altered, which is mirrored in genomic loci bound by Sox2/Oct4 complexes.

Lac repressor, the first discovered transcriptional regulator, has been shown to confer multiple-modes of binding to its operator sites depending on the central spacer length. Other homolog members in the LacI/GalR family (PurR and YcjW) cannot bind their operator sites with similar structural flexibility. To decipher the underlying mechanism for this unique property, we used Spec-seq approach combined with site-directed mutagenesis to quantify the DNA binding specificity of multiple hybrids of lacI and PurR. We find that lac repressor’s recognition di-residues YQ and its hinge helix loop regions are both critical for its structural flexibility. Also, specificity profiling of the whole lac operator suggests that a simple additive model from single variants suffice to predict other multivariant sites’ energy reasonably well, and the genome occupancy model based on this specificity data correlates well with in vivo lac repressor binding profile.