Reciprocating RNA Polymerase batters through roadblocks.

RNA polymerases must transit through protein roadblocks to produce full-length transcripts. Here we report real-time measurements of Escherichia coli RNA polymerase passing through different barriers. As intuitively expected, assisting forces facilitated, and opposing forces hindered, RNA polymerase passage through lac repressor protein bound to natural binding sites. Force-dependent differences were significant at magnitudes as low as 0.2 pN and were abolished in the presence of the transcript cleavage factor GreA, which rescues backtracked RNA polymerase. In stark contrast, opposing forces promoted passage when the rate of RNA polymerase backtracking was comparable to, or faster than the rate of dissociation of the roadblock, particularly in the presence of GreA. Our experiments and simulations indicate that RNA polymerase may transit after roadblocks dissociate, or undergo cycles of backtracking, recovery, and ramming into roadblocks to pass through. We propose that such reciprocating motion also enables RNA polymerase to break protein-DNA contacts that hold RNA polymerase back during promoter escape and RNA chain elongation. This may facilitate productive transcription in vivo.

Engineering and application of LacI mutants with stringent expressions.

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the Plac /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS).

A tuneable genetic switch for tight control of tac promoters in Escherichia coli boosts expression of synthetic injectisomes.

Biosafety of engineered bacteria as living therapeutics requires a tight regulation to control the specific delivery of protein effectors, maintaining minimum leakiness in the uninduced (OFF) state and efficient expression in the induced (ON) state. Here, we report a three repressors (3R) genetic circuit that tightly regulates the expression of multiple tac promoters (Ptac) integrated in the chromosome of E. coli and drives the expression of a complex type III secretion system injectisome for therapeutic protein delivery. The 3R genetic switch is based on the tetracycline repressor (TetR), the non-inducible lambda repressor cI (ind-) and a mutant lac repressor (LacIW220F ) with higher activity. The 3R switch was optimized with different protein translation and degradation signals that control the levels of LacIW220F . We demonstrate the ability of an optimized switch to fully repress the strong leakiness of the Ptac promoters in the OFF state while triggering their efficient activation in the ON state with anhydrotetracycline (aTc), an inducer suitable for in vivo use. The implementation of the optimized 3R switch in the engineered synthetic injector E. coli (SIEC) strain boosts expression of injectisomes upon aTc induction, while maintaining a silent OFF state that preserves normal growth in the absence of the inducer. Since Ptac is a commonly used promoter, the 3R switch may have multiple applications for tight control of protein expression in E. coli. In addition, the modularity of the 3R switch may enable its tuning for the control of Ptac promoters with different inducers.

Detecting DNA Loops Using Tethered Particle Motion.

The range of motion of a micron-sized bead tethered by a single polymer provides a dynamic readout of the effective length of the polymer. The excursions of the bead may reflect the intrinsic flexibility and/or topology of the polymer as well as changes due to the action activity of ligands that bind the polymer. This is a simple yet powerful experimental approach to investigate such interactions between DNA and proteins as demonstrated by experiments with the lac repressor. This protein forms a stable, tetrameric oligomer with two binding sites and can produce a loop of DNA between recognition sites separated along the length of a DNA molecule.

Simulated pressure changes in LacI suggest a link between hydration and functional conformational changes.

The functions of many proteins are associated with interconversions among conformational substates. However, these substates can be difficult to measure experimentally, and determining contributions from hydration changes can be especially difficult. Here, we assessed the use of pressure perturbations to sample the substates accessible to the Escherichia coli lactose repressor protein (LacI) in various liganded forms. In the presence of DNA, the regulatory domain of LacI adopts an Open conformation that, in the absence of DNA, changes to a Closed conformation. Increasing the simulation pressure prevented the transition from an Open to a Closed conformation, in a similar manner to the binding of DNA and anti-inducer, ONPF. The results suggest the hydration of specific residues play a significant role in determining the population of different LacI substates and that simulating pressure perturbation could be useful for assessing the role of hydration changes that accompany functionally-relevant amino acid substitutions.

Imaging of genomic loci in Trypanosoma brucei using an optimised LacO-LacI system.

Visualisation of genomic loci by microscopy is essential for understanding nuclear organisation, particularly at the single cell level. One powerful technique for studying the positioning of genomic loci is through the Lac Operator-Lac Repressor (LacO-LacI) system, in which LacO repeats introduced into a specific genomic locus can be visualised through expression of a LacI-protein fused to a fluorescent tag. First utilised in Trypanosoma brucei over 20 years ago, we have now optimised this system with short, stabilised LacO repeats of less than 2 kb paired with a constitutively expressed mNeongreen::LacI fusion protein to facilitate visualisation of genomic loci. We demonstrate the compatibility of this system with super-resolution microscopy and propose its suitability for multiplexing with inducible RNAi or protein over expression which will allow analysis of nuclear organisation after perturbation of gene expression.

Genetic switching by the Lac repressor is based on two-state Monod-Wyman-Changeux allostery.

High-resolution NMR spectroscopy enabled us to characterize allosteric transitions between various functional states of the dimeric Escherichia coli Lac repressor. In the absence of ligands, the dimer exists in a dynamic equilibrium between DNA-bound and inducer-bound conformations. Binding of either effector shifts this equilibrium toward either bound state. Analysis of the ternary complex between repressor, operator DNA, and inducer shows how adding the inducer results in allosteric changes that disrupt the interdomain contacts between the inducer binding and DNA binding domains and how this in turn leads to destabilization of the hinge helices and release of the Lac repressor from the operator. Based on our data, the allosteric mechanism of the induction process is in full agreement with the well-known Monod-Wyman-Changeux model.

Ligand-specific changes in conformational flexibility mediate long-range allostery in the lac repressor.

Biological regulation ubiquitously depends on protein allostery, but the regulatory mechanisms are incompletely understood, especially in proteins that undergo ligand-induced allostery with few structural changes. Here we used hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to map allosteric effects in a paradigm ligand-responsive transcription factor, the lac repressor (LacI), in different functional states (apo, or bound to inducer, anti-inducer, and/or DNA). Although X-ray crystal structures of the LacI core domain in these states are nearly indistinguishable, HDX/MS experiments reveal widespread differences in flexibility. We integrate these results with modeling of protein-ligand-solvent interactions to propose a revised model for allostery in LacI, where ligand binding allosterically shifts the conformational ensemble as a result of distinct changes in the rigidity of secondary structures in the different states. Our model provides a mechanistic basis for the altered function of distal mutations. More generally, our approach provides a platform for characterizing and engineering protein allostery.

Silencing Transcription from an Influenza Reverse Genetics Plasmid in E. coli Enhances Gene Stability.

Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics. Here, we demonstrate that genes encoding the influenza surface antigens hemagglutinin and neuraminidase have varying stability when cloned into a common RG plasmid and transformed into Escherichia coli. Using GFP as a reporter, we demonstrate that E. coli expresses the target genes in the RG plasmid at low levels. Incorporating lac operators or a transcriptional terminator into the plasmid reduced expression and stabilized the viral genes to varying degrees. Sandwiching the viral gene between two lac operators provided the largest contribution to stability and we confirmed the stabilization is Lac repressor-dependent and crucial for subsequent plasmid propagations in E. coli. Viruses rescued from the lac operator-stabilized plasmid displayed similar kinetics and titers to the original plasmid in two different viral backbones. Together, these results indicate that silencing transcription from the plasmid in E. coli helps to maintain the correct influenza gene sequence and that the lac operator addition does not impair virus production. It is envisaged that sandwiching DNA segments between lac operators can be used for reducing DNA segment instability in any plasmid that is propagated in E. coli which express the Lac repressor.

Testing the Effect of Histone Acetyltransferases on Local Chromatin Compaction.

Experiments determining the chromatin association of histone acetylases (HATs) and deacetylases (HDACs) at the genome-wide level provide precise maps of locus occupancy, but do not allow conclusions on the functional consequences of this locus-specific enrichment. Here we describe a protocol that allows tethering of HATs or HDACs to specific genomic loci upon fusion with a fluorescent protein and a DNA-binding protein such as the E. coli Lac repressor (LacI), which binds to genomically inserted lac operon sequences (lacO) via DNA/protein interactions. Integration of these lacO sequences into a genomic region of interest allows to monitor the functional consequences of HAT/HDAC targeting on chromatin (de)compaction, histone modification, and interaction with other proteins by quantitative light microscopy, as described here. As DNA-binding of LacI can be tightly controlled by the addition of galactose-derivatives, this method also allows to monitor the effects of locus-specific recruitment in a time-resolved manner.

A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios.

The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild-type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2, and VP3, from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires a theoretical modification of the translational context. We have developed a Lac repressor-inducible system to empirically regulate the expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer, and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that the ratios of capsid proteins expressed were different than the ratios that ultimately were in purified capsids. Overexpressed VP1 did not become incorporated into capsids, while overexpressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling the production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.

Conformational Change of Transcription Factors from Search to Specific Binding: A lac Repressor Case Study.

In a process known as facilitated diffusion, DNA-binding proteins find their target sites by combining three-dimensional diffusion and one-dimensional scanning of the DNA. Following the trade-off between speed and stability, agile exploration of DNA requires loose binding, whereas, at the DNA target site, the searching protein needs to establish tight interactions with the DNA. To enable both efficient search and stable binding, DNA-binding proteins and DNA often switch conformations upon recognition. Here, we study the one-dimensional diffusion and DNA binding of the dimeric lac repressor (LacI), which was reported to adopt two different conformations when binding different conformations of DNA. Using coarse-grained molecular dynamic simulations, we studied the diffusion and the sequence-specific binding of these conformations of LacI, as well as their truncated or monomeric variants, with two DNA conformations: straight and bent. The simulations were compared to experimental observables. This study supports that linear diffusion along DNA combines tight rotation-coupled groove tracking and rotation-decoupled hopping, where the protein briefly dissociates and reassociates just a few base pairs away. Tight groove tracking is crucial for target-site recognition, while hopping speeds up the overall search process. We investigated the diffusion of different LacI conformations on DNA and show how the flexibility of LacI's hinge regions ensures agility on DNA as well as faithful groove tracking. If the hinge regions instead form α-helices at the protein-DNA interface, tight groove tracking is not possible. On the contrary, the helical hinge region is essential for tight binding to bent, specific DNA, for the formation of the specific complex. Based on our study of different encounter complexes, we argue that the conformational change in LacI and DNA bending are somewhat coupled. Our findings underline the importance of two distinct protein conformations for facilitated diffusion and specific binding, respectively.

Robustness of DNA looping across multiple cell divisions in individual bacteria.

DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.

Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops.

Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, 400 bp LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible for this reduction in pause time. In support of this idea, RNA polymerase transcribed 6-fold more slowly through looped DNA and paused at LacI obstacles for 66% less time on positively supercoiled compared to relaxed templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase facilitated elongation along topologically complex, protein-coated templates.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation.

BACKGROUND: The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host. RESULTS: Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels. CONCLUSIONS: The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

Sequence specificity in DNA binding is mainly governed by association.

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.

Switching off: The phenotypic transition to the uninduced state of the lactose uptake pathway.

The lactose uptake pathway of E. coli is a paradigmatic example of multistability in gene regulatory circuits. In the induced state of the lac pathway, the genes comprising the lac operon are transcribed, leading to the production of proteins that import and metabolize lactose. In the uninduced state, a stable repressor-DNA loop frequently blocks the transcription of the lac genes. Transitions from one phenotypic state to the other are driven by fluctuations, which arise from the random timing of the binding of ligands and proteins. This stochasticity affects transcription and translation, and ultimately molecular copy numbers. Our aim is to understand the transition from the induced to the uninduced state of the lac operon. We use a detailed computational model to show that repressor-operator binding and unbinding, fluctuations in the total number of repressors, and inducer-repressor binding and unbinding all play a role in this transition. Based on the timescales on which these processes operate, we construct a minimal model of the transition to the uninduced state and compare the results with simulations and experimental observations. The induced state turns out to be very stable, with a transition rate to the uninduced state lower than 2×10-9 per minute. In contrast to the transition to the induced state, the transition to the uninduced state is well described in terms of a 2D diffusive system crossing a barrier, with the diffusion rates emerging from a model of repressor unbinding.

Modular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity.

At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). These sensors have a modular design that should generalize to other bacterial signaling systems in the future.

Biological innovation in the functional landscape of a model regulator, or the lactose operon repressor.

The operon model was proposed six decades ago. And yet, despite all this time, the lactose operon repressor, LacI, remains a subject of major interest. While it is well established that LacI can exist in two functional forms, one that renders the operon inactive via binding of LacI to DNA and another, bound to an inducer that does not allow repression, how it switches from one to the other is still not well understood. The construction of a library of several tens of thousands of LacI mutants has revealed some unexpected features. In particular, the transition implemented in some of them reveals a new type of transcription regulation: band-pass (OFF/ON/OFF) and band-stop (ON/OFF/ON) filters. This makes it natural to think that it is the network of hydrogen bonds associated with the water bound to the molecule that allows the remote interconnection between the binding site to an inducer molecule and the one that binds it to the DNA.

Le modèle de l'opéron a été proposé il y a six décennies. Et pourtant, malgré tout ce temps passé, le répresseur de l'opéron lactose, LacI, reste un sujet d'intérêt majeur. S'il est bien établi que LacI peut exister sous deux formes fonctionnelles, l'une qui rend inactif l'opéron via la liaison de LacI à l'ADN et l'autre, liée à un inducteur qui ne permet pas cette répression, la façon dont il passe de l'une à l'autre n'est toujours pas bien comprise. La construction d'une bibliothèque de plusieurs dizaines de milliers de mutants de LacI a mis au jour des caractéristiques inattendues. En particulier la transition mise en œuvre dans certains d'entre eux fait émerger un nouveau type de régulation de la transcription : filtre à bande passante (INACTIF/ACTIF/INACTIF) et filtre à bande d'arrêt (ACTIF/INACTIF/ACTIF). Il est naturel de penser que c'est le réseau des liaisons hydrogène associées à l'eau liée à la molécule qui permet l'interconnexion à distance entre le site de liaison à une molécule inductrice et celui qui le lie à l'ADN.

Rheostat functional outcomes occur when substitutions are introduced at nonconserved positions that diverge with speciation.

When amino acids vary during evolution, the outcome can be functionally neutral or biologically-important. We previously found that substituting a subset of nonconserved positions, "rheostat" positions, can have surprising effects on protein function. Since changes at rheostat positions can facilitate functional evolution or cause disease, more examples are needed to understand their unique biophysical characteristics. Here, we explored whether "phylogenetic" patterns of change in multiple sequence alignments (such as positions with subfamily specific conservation) predict the locations of functional rheostat positions. To that end, we experimentally tested eight phylogenetic positions in human liver pyruvate kinase (hLPYK), using 10-15 substitutions per position and biochemical assays that yielded five functional parameters. Five positions were strongly rheostatic and three were non-neutral. To test the corollary that positions with low phylogenetic scores were not rheostat positions, we combined these phylogenetic positions with previously-identified hLPYK rheostat, "toggle" (most substitution abolished function), and "neutral" (all substitutions were like wild-type) positions. Despite representing 428 variants, this set of 33 positions was poorly statistically powered. Thus, we turned to the in vivo phenotypic dataset for E. coli lactose repressor protein (LacI), which comprised 12-13 substitutions at 329 positions and could be used to identify rheostat, toggle, and neutral positions. Combined hLPYK and LacI results show that positions with strong phylogenetic patterns of change are more likely to exhibit rheostat substitution outcomes than neutral or toggle outcomes. Furthermore, phylogenetic patterns were more successful at identifying rheostat positions than were co-evolutionary or eigenvector centrality measures of evolutionary change.