Ligand-induced changes in dynamics mediate long-range allostery in the lac repressor

Allostery, broadly defined as a protein's functional response to distal perturbations, is fundamental to biological regulation. In classical models, allosteric ligand binding produces a defined set of structural changes in the protein, resulting in a different low-energy conformation. Proteins that undergo ligand-induced allostery with few observable structural changes therefore frustrate interpretations by classical models. Here we used hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to map the allosteric effects in a paradigm ligand-responsive allosteric transcription factor, the lac repressor (LacI). X-ray crystal structures of the core domain of LacI bound to different small molecule ligands, or the DNA operator, show less than 1.5 Å difference in the protein all-atom root-mean-square-deviation (RMSD) between any two structures. Despite this high degree of similarity among static structures, our HDX/MS experiments reveal widespread and unexpected differences in the flexibility of secondary structures in the LacI core domain in each functional state. We propose a model in which ligand binding allosterically switches the functional response of the repressor by selectively changing the dynamics of particular secondary structure elements relative to each other, shifting the conformational ensemble of the protein between mutually incompatible DNA-bound and inducer-bound states. Our model also provides a mechanistic context for the altered functions of thousands of documented LacI mutants. Furthermore, our approach provides a platform for characterizing and engineering allosteric responses in proteins.

Proteins mediating different DNA topologies block RNAP elongation with different efficiency

Many DNA-binding proteins induce topological structures such as loops or wraps through binding to two or more sites along the DNA. Such topologies may regulate transcription initiation and may also be roadblocks for elongating RNA polymerase (RNAP). Remarkably, a lac repressor protein bound to a weak binding site (O2) does not obstruct RNAP in vitro but becomes an effective roadblock when securing a loop of 400 bp between two widely separated binding sites. To investigate whether topological structures mediated by proteins bound to closely spaced binding sites and interacting cooperatively also represent roadblocks, we compared the effect of the lambda CI and 186 CI repressors on RNAP elongation. Dimers of lambda CI can bind to two sets of adjacent sites separated by hundreds of bp and form a DNA loop via the interaction between their C-terminal domains. The 186 CI protein can form a wheel of seven dimers around which specific DNA binding sequences can wrap. Atomic force microscopy (AFM) was used to image transcription elongation complexes of DNA templates that contained binding sites for either the lambda or 186 CI repressor. While RNAP elongated past lambda CI on unlooped DNA, as well as past 186 CI-wrapped DNA, it did not pass the lambda CI-mediated loop. These results may indicate that protein-mediated loops with widely separated binding sites more effectively block transcription than a wrapped topology with multiple, closely spaced binding sites.

Designed architectural proteins that tune DNA looping in bacteria

Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

Designed architectural proteins that tune DNA looping in bacteria

Architectural proteins alter the shape of DNA, often by distorting the double helix and introducing sharp kinks that relieve strain in tightly-bent DNA structures. Here we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from E. coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

Deep representation learning improves prediction of LacI-mediated transcriptional repression

Recent progress in DNA synthesis and sequencing technology has enabled systematic studies of protein function at a massive scale. We explore a deep mutational scanning study that measured the transcriptional repression function of 43,669 variants of the Escherichia coli LacI protein. We analyze structural and evolutionary aspects that relate to how the function of this protein is maintained, including an in-depth look at the C-terminal domain. We develop a deep neural network to predict transcriptional repression mediated by the lac repressor of Escherichia coli using experimental measurements of variant function. When measured across 10 separate training and validation splits using 5,009 single mutations of the lac repressor, our best-performing model achieved a median Pearson correlation of 0.79, exceeding any previous model. We demonstrate that deep representation learning approaches, first trained in an unsupervised manner across millions of diverse proteins, can be fine-tuned in a supervised fashion using lac repressor experimental datasets to more effectively predict a variant’s effect on repression. These findings suggest a deep representation learning model may improve the prediction of other important properties of proteins.

Introduction to a special issue of <i>Magnetic Resonance</i> in honour of Robert Kaptein at the occasion of his 80th birthday

This publication, in honour of Robert Kaptein's 80th birthday, contains contributions from colleagues, many of whom have worked with him, and others who admire his work and have been stimulated by his research. The contributions show current research in biomolecular NMR, spin hyperpolarisation and spin chemistry, including CIDNP (chemically induced dynamic nuclear polarisation), topics to which he has contributed enormously. His proposal of the radical pair mechanism was the birth of the field of spin chemistry, and the laser CIDNP NMR experiment on a protein was a major breakthrough in hyperpolarisation research. He set milestones for biomolecular NMR by developing computational methods for protein structure determination, including restrained molecular dynamics and 3D NMR methodology. With a lac repressor headpiece, he determined one of the first protein structures determined by NMR. His studies of the lac repressor provided the first examples of detailed studies of protein nucleic acid complexes by NMR. This deepened our understanding of protein DNA recognition and led to a molecular model for protein sliding along the DNA. Furthermore, he played a leading role in establishing the cluster of NMR large-scale facilities in Europe. This editorial gives an introduction to the publication and is followed by a biography describing his contributions to magnetic resonance.

Transcription elongation through lac repressor-mediated DNA loops

During elongation, RNA polymerase (RNAP) must navigate through proteins that decorate genomic DNA. Several of these mediate long-distance interactions via structures, such as loops, that alter DNA topology and create torsional barriers. We used the tethered particle motion (TPM) technique and magnetic tweezers to monitor transcription of DNA templates in the presence of the lac repressor (LacI) protein which could bind at two sites, one proximal to, and one distal from, the promoter. The bivalent LacI tetramer binds recognition sites (operators) with up to nanomolar affinity depending on the sequence, and the concentration of LacI was adjusted to promote binding to either one or both operators, so as to produce unlooped or looped DNA. We observed that RNAP pausing before a LacI-securing loop was determined not by the affinity of LacI for the operator, but by the order in which the elongating RNAP encountered these operators. TPM experiments showed that, independent of affinity, LacI bound at the promoter-proximal operator became a stronger roadblock when securing a loop. In contrast, LacI bound to the distal operator was a weaker roadblock in a looped configuration suggesting that RNAP might more easily displace LacI obstacles within a torsion-constrained DNA loop. Since protein junctions can efficiently block the diffusion of DNA supercoiling, these data indicate that the positive supercoiling generated ahead of a transcribing RNAP may facilitate the dissociation of a roadblock. In support of this idea, magnetic tweezers measurements indicated that pauses are shorter when RNAP encounters obstacles on positively supercoiled than on relaxed DNA. Furthermore, at similar winding levels of the DNA template, RNAP pause duration decreased with tension. These findings are significant for our understanding of transcription within the crowded and tensed nucleoid.

Negative DNA supercoiling tunes protein-mediated looping

Protein-mediated DNA looping is a fundamental mechanism of gene regulation. Such loops occur stochastically, and a calibrated response to environmental stimuli would seem to require more deterministic behavior, so experiments were preformed to determine whether additional proteins and/or DNA supercoiling might be definitive. In experiments on DNA looping mediated by the Escherichia coli lac repressor protein, increasing compaction by the nucleoid-associated protein, HU, progressively increased the average looping probability for an ensemble of single molecules. Despite this trend, the looping probabilities associated with individual molecules ranged from 0 to 100 throughout the titration, and observations of a single molecule for an hour or longer were required to observe the statistical looping behavior of the ensemble, ergodicity. Increased negative supercoiling also increased the looping probability for an ensemble of molecules, but the looping probabilities of individual molecules more closely resembled the ensemble average. Furthermore, supercoiling accelerated the loop dynamics such that in as little as twelve minutes of observation most molecules exhibited the looping probability of the ensemble. Notably, this is within the timescale of the doubling time of the bacterium. DNA supercoiling, an inherent feature of genomes across kingdoms, appears to be a fundamental determinant of time-constrained, emergent behavior in otherwise random molecular activity.

Transcriptional analysis reveals key insights into seasonal induced anthocyanin degradation and leaf color transition in purple tea (Camellia sinensis (L.) O. Kuntze)

Purple-tea, an anthocyanin rich cultivar has recently gained popularity due to its health benefits and captivating leaf appearance. However, the sustainability of purple pigmentation and anthocyanin content during production period is hampered by seasonal variation. To understand seasonal dependent anthocyanin pigmentation in purple tea, global transcriptional and anthocyanin profiling was carried out in tea shoots with two leaves and a bud harvested during in early (reddish purple: S1_RP), main (dark gray purple: S2_GP) and backend flush (moderately olive green: S3_G) seasons. Of the three seasons, maximum accumulation of total anthocyanin content was recorded in S2_GP, while least amount was recorded during S3_G. Reference based transcriptome assembly of 412 million quality reads resulted into 71,349 non-redundant transcripts with 6081 significant differentially expressed genes. Interestingly, key DEGs involved in anthocyanin biosynthesis [PAL, 4CL, F3H, DFR and UGT/UFGT], vacuolar trafficking [ABC, MATE and GST] transcriptional regulation [MYB, NAC, bHLH, WRKY and HMG] and Abscisic acid signaling pathway [PYL and PP2C] were significantly upregulated in S2_GP. Conversely, DEGs associated with anthocyanin degradation [Prx and lac], repressor TFs and key components of auxin and ethylene signaling pathways [ARF, AUX/IAA/SAUR, ETR, ERF, EBF1/2] exhibited significant upregulation in S3_G, correlating positively with reduced anthocyanin content and purple coloration. The present study for the first-time elucidated genome-wide transcriptional insights and hypothesized the involvement of anthocyanin biosynthesis activators/repressor and anthocyanin degrading genes via peroxidases and laccases during seasonal induced leaf color transition in purple tea. Futuristically, key candidate gene(s) identified here can be used for genetic engineering and molecular breeding of seasonal independent anthocyanin-rich tea cultivars.

Characterization of gene repression by designed transcription activator-like effector dimer proteins

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the E. coli lac operon switch, where DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed Transcription Activator-Like Effector Dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system.Statement of SignificanceWe are designing and testing in living bacteria artificial DNA looping proteins engineered based on principles learned from studies of the E. coli lac repressor. The engineered proteins are based on artificial dimers of Transcription Activator-Like Effector (TALE) proteins that have programmable DNA binding specificities. The current work is the first to create unique DNA repression loops using this approach. Systematic study of repression as a function of loop size, with data fitting to a thermodynamic model, now allows this system to be compared in detail with lac repressor loops, and relevant biophysical parameters to be estimated. This approach has implications for the artificial regulation of gene expression.