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

Abstract

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...

Deciphering the protein‐DNA code of bacterial winged helix‐turn‐helix transcription factors

“…In this format, highamplitude positions contribute strongly to specificity, with bases above and below the center-line increasing and reducing affinity, respectively. reaction [51,53], in contrast to other techniques that may incur experimental or computational artefacts [55]. Using Spec-seq, we found no evidence of the asymmetric halfsite recognition previously observed for RstA using SELEX ( Figure 4C, lower).…”

“…Bound and unbound DNA bands were extracted from lanes indicated with an asterisk, and relative free energies of binding to different DNA sequences were calculated by Spec-seq [45,53]. Energy logos depicting relative free energies for each nucleotide in the binding site [54] are located below their corresponding gel, with gray bars on the y-axis are normalized to a magnitude of +/-1 in units of ( -kT) across all logos.…”

Lactose repressor hinge domain independently binds DNA

“…Unlike other members of this family, LacI is both tetrameric and capable of binding to a variety of semi‐symmetric operator sites that differ in the length of the central spacer sequence . Binding is achieved by an asymmetric arrangement of the two helix‐turn‐helix motifs within the dimeric DNA binding unit of the tetramer . Two features of LacI are required for this flexible recognition process: (i) the hinge helix, which recognizes and kinks the central region of the operator in a symmetric target site, and (ii) a YQ sequence at positions 17–18 in the N‐terminal DNA binding domain that recognizes sequences in the flanking regions of the operator DNA sequence .…”

Transcription factor–DNA binding: beyond binding site motifs

“…SELEX has been adapted for characterizing the binding of TF pairs and complexes [41,43]. Spec-seq is similar to SELEX-seq but can accurately measure relative DNA binding affinities and is well suited for assessing the impacts of DNA sequence variants [44]. Genomic-context PBMs (gcPBMs) interrogate select 30- to 36-bp sequences and have elucidated the contributions of flanking genomic sequences to motif recognition [38].…”