Lac Repressor Protein Research Articles

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.

Proteins mediating different DNA topologies block RNAP elongation with different efficiency

Most DNA-binding proteins alter the conformation of the double helix and many induce topological structures such as loops or wraps by binding to two or more sites along the DNA. While such topologies may efficiently regulate initiation at a promoter, they may also be roadblocks for an incoming RNA polymerase (RNAP) transcribing a different gene. The lac repressor protein bound to a weak binding site was shown to turn into a strong roadblock when securing a loop of 400 bp. The question remains if other proteins could similarly become strong roadblocks when mediating different types of topological structures.

Escherichia coli \u03c370 promoters allow expression rate control at the cellular level in genome-integrated expression systems

BackgroundThe genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences.ResultsWe showed that, in genome-integrated E. coli expression systems that used σ70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacIQ, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.ConclusionsBased on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

Transcriptional Obstacles Modify Repetitive Elongation

Transcription is regulated by multiple factors including those in the path of RNA polymerase that can block elongation. Obstacles were produced using the lac repressor protein (LacI) binding to the high affinity O1 sequence in DNA tethers anchored by RNA polymerase at one end. Passage through LacI obstacles was monitored using either tethered particle motion (TPM) without tension on the DNA tether, or magnetic tweezers (MTs) applying sub-piconewton tension. When E. coli RNAP labeled on either the α or β’ subunits encountered LacI, it paused and the tether length remained constant for up to minutes. However, three peculiar behaviors were noted. (i) In the absence of tension, the pausing of RNAPs with either labeling was comparable. In contrast, while RNAP attached to glass via hemagglutinin antigen tags on α subunits paused for longer at LacI when tension opposed rather than assisted transcriptional translocation, RNAP attached to beads via a biotin-labeled β’ subunit paused more briefly at LacI when tension opposed rather than assisted transcriptional translocation. (ii) After pausing at a LacI obstacle with tension opposed to transcription, RNAP often abruptly re-commenced elongation from the promoter. Occasionally, having surpassed an obstacle and reached the end of the template, RNAP abruptly translocated to re-commence transcription from the obstacle. (iii) Occasionally, after reaching the end of the template, RNAPs elongating under assisting tension switched strands to transcribe in the opposite direction against tension opposing transcriptional translocation. Although forces applied to different subunits of the enzyme certainly influenced activity, RNAP translocated rapidly or conserved an interaction with the promoter or an obstacle to perform repeated cycles of elongation. Complete reversal of the direction of elongation occurred at double-stranded ends. Repetition and reversal expand the gamet of transcriptional output.

The lac repressor hinge helix in context: The effect of the DNA binding domain and symmetry

The Lac system of genes has been an important model system in understanding gene regulation. When the dimer lac repressor protein binds to the correct DNA sequence, the hinge region of the protein goes through a disorder to order transition. The hinge region is disordered when binding to nonoperator sequences. This region of the protein must pay a conformational entropic penalty to order when it is bound to operator DNA. Structural studies show that this region is flexible. Previous simulations showed that this region is disordered when free in solution without the DNA binding domain present. Our simulations corroborate that this region is extremely flexible in solution, but we find that the presence of the DNA binding domain proximal to the hinge helix and salt make the ordered conformation more favorable even without DNA present.

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System.

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

Investigating Structure and Topology of Protein-Mediated DNA Loops via Computational Modeling of Elastic Energy

Many biological processes involve deformations of DNA 3D structure. DNA looping generated by a protein bound to two distant sites on the double helix is a familiar mechanism employed in regulation of cellular processes. We employ a computational method to obtain minimum energy structures of DNA loops mediated by the Lac repressor protein, whereby the potential energy of elastic deformation is optimized. By evaluating the energy required to deform DNA, we can predict the likely spatial pathway of the DNA strand. By adjusting the optimization parameters, we can evaluate the effects of torsional stress on loop configuration and global topology. For instance, altering the position or orientation of the end constraints provides insight into mechanisms that proteins may use to control DNA folding. Alternatively, changing the chain length of the constrained DNA molecule alters the total twist, which is reflected in the supercoiling of the molecule. Adjusting the intrinsic helical repeat in the optimization simulates changes in the cellular environment such as found with increased salt content. Using knowledge of the cost of deforming base-pair steps of different types we simulate the effect of nucleotide sequence on the overall structure of constrained DNA chains.

Price of disorder in the lac repressor hinge helix.

The Lac system of genes has been pivotal in understanding gene regulation. When the lac repressor protein binds to the correct DNA sequence, the hinge region of the protein goes through a disorder to order transition. The structure of this region of the protein is well understood when it is in this bound conformation, but less so when it is not. Structural studies show that this region is flexible. Our simulations show this region is extremely flexible in solution; however, a high concentration of salt can help kinetically trap the hinge helix. Thermodynamically, disorder is more favorable without the DNA present.

Salt Effects on Protein Folding Thermodynamics.

Salts differ in their ability to stabilize protein conformations, thereby affecting the thermodynamics and kinetics of protein folding. We developed a coarse-grained protein model that can predict salt-induced changes in protein properties by using the transfer free-energy data of various chemical groups from water to salt solutions. Using this model and molecular dynamics simulations, we probed the effect of seven different salts on the folding thermodynamics of the DNA binding domain of lac repressor protein ( lac-DBD) and N-terminal domain of ribosomal protein (NTL9). We show that a salt can act as a protein stabilizing or destabilizing agent depending on the protein sequence and folded state topology. The computed thermodynamic properties, especially the m values for various salts, which reveal the relative ability of a salt to stabilize the protein folded state, are in quantitative agreement with the experimentally measured values. The computations show that the degree of protein compaction in the denatured ensemble strongly depends on the salt identity, and for the same variation in salt concentration, the compaction in the protein dimensions varies from ∼4% to ∼30% depending on the salt. The transition-state ensemble (TSE) of lac-DBD is homogeneous and polarized, while the TSE of NTL9 is heterogeneous and diffusive. Salts induce subtle structural changes in the TSE that are in agreement with Hammond's postulate. The barrier to protein folding tends to disappear in the presence of moderate concentrations (∼3-4 m) of strongly stabilizing salts.

Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching.

In any living cell, genome maintenance is carried out by DNA-binding proteins that recognize specific sequences among a vast amount of DNA. This includes fundamental processes such as DNA replication, DNA repair, and gene expression and regulation. Here, we study the mechanism of DNA target search by a single lac repressor protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and few base-pair resolution technique based on laser tweezers. We measure 1D-diffusion of proteins on DNA at physiological salt concentrations with 20 bp resolution and find that sliding of LacI along DNA is sequence dependent. We show that only allosterically activated LacI slides along non-specific DNA sequences during target search, whereas the inhibited conformation does not support sliding and weakly interacts with DNA. Moreover, we find that LacI undergoes a load-dependent conformational change when it switches between sliding and strong binding to the target sequence. Our data reveal how DNA sequence and molecular switching regulate LacI target search process and provide a comprehensive model of facilitated diffusion for LacI.

Development of cyclic AMP receptor protein-based artificial transcription factor for intensifying gene expression.

Vector-dependent gene overexpression typically relies on an efficient operon and sufficient RNA polymerases (RNAPs). The lac (lactose) operon is a paradigm of transcription control, and cyclic AMP receptor protein (CRP) is a global regulator capable of recruiting RNAPs. However, the gap between lac operon and CRP has not been well bridged. In this work, CRP was fused to lac repressor protein (lacI) to form an artificial transcription factor (ATF) with the expectation that when LacI acted on the lacO-positioned upstream of gene of interest, the LacI-tethered CRP would trap RNAPs and thus improve the expression of PuuC, an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde (3-HPA) to 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae. As expected, SDS-PAGE and HPLC showed enhanced PuuC expression and 3-HP production, respectively, compared to the control strain without expressing chimeric protein LacI-CRP. Moreover, quantitative real-time PCR demonstrated increased transcription levels of both PuuC and RNAP coding genes. In shake-flask cultivation, the recombinant K. pneumoniae strain coexpressing LacI-CRP and PuuC produced 1.67-fold of 3-HP relative to the stain only overexpressing PuuC. In bioreactor cultivation, the strain coexpressing LacI-CRP and PuuC produced 35.1g/L 3-HP, whereas the strain without expressing LacI-CRP generated only 9.8g/L 3-HP. Overall, these results indicated that as an ATF, LacI-CRP significantly boosted PuuC expression and 3-HP production. We envision that LacI-CRP as a plug-and-play part can be used for regulating gene expression.

Understanding radiation damage on sub-cellular scale using RADAMOL simulation tool

We present an overview of the biophysical model RADAMOL developed as a Monte Carlo simulation tool for physical, physico-chemical and chemical stages of ionizing radiation action. Direct and indirect radiation damage by 10keV electrons, and protons and alpha particles with energies from 1MeV up to 30MeV to a free DNA oligomer or DNA in the complex with lac repressor protein is analyzed. The role of radiation type and energy, oxygen concentration and DNA interaction with proteins on yields and distributions of primary biomolecular damage is demonstrated and discussed.

Insights into Genome Architecture Deduced from the Properties of Short Lac Repressor-mediated DNA Loops.

Genomic DNA is vastly longer than the space allotted to it in a cell. The molecule must fold with a level of organization that satisfies the imposed spatial constraints as well as allows for the processing of genetic information. Key players in this organization include the negative supercoiling of DNA, which facilitates the unwinding of the double-helical molecule, and the associations of DNA with proteins, which partition the DNA into isolated loops, or domains. In order to gain insight into the principles of genome organization and to visualize the folding of spatially constrained DNA, we have developed new computational methods to identify the preferred three-dimensional pathways of protein-mediated DNA loops and to characterize the topological properties of these structures. Here we focus on the levels of supercoiling and the spatial arrangements of DNA in model nucleoprotein systems with two topological domains. We construct these systems by anchoring DNA loops in opposing orientations on a common protein-DNA assembly, namely the Lac repressor protein with two bound DNA operators. The linked pieces of DNA form a covalently closed circle such that the protein attaches to two widely spaced sites along the DNA. We examine the effects of operator spacing, loop orientation, and long-range contacts on overall chain configuration and topology and discuss our findings in the context of classic experiments on the effects of supercoiling and operator spacing on Lac repressor-mediated looping and recent work on the role of proteins as barriers that divide genomes into independent topological domains.

The Hinge Region Strengthens the Nonspecific Interaction between Lac-Repressor and DNA: A Computer Simulation Study.

LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex.

HU Protein and DNA Supercoiling Dramatically Enhance Lac-Repressor-Mediated DNA Looping

DNA loops mediated by proteins that bind with different affinity to distant sites regulate multiple aspects of DNA metabolism, such as transcription, replication and recombination. Binding of accessory proteins and DNA supercoiling are two factors that affect the formation of DNA loops. Previous studies indicate that the heat unstable protein (HU) enhances the formation of short loops perhaps by changing the flexibility of DNA upon binding non-specifically. However, for loops long enough such that DNA stiffness is not limiting, the effect of HU on loop formation is not well understood. Negative supercoiling also enhances the formation of long loops. To better understand how binding affinity, loop length, HU and negative supercoiling affect looping, single molecule experiments were performed with the lac repressor protein (LacI) as a DNA looping protein. Using tethered particle motion, a titration of the formation of Lac repressor mediated-loop was carried out using three different DNA templates, OID-900-O1, OID-400-O1, and O1-400-O2 (Ostrong-loop size (bp)-Oweak). It showed that long loops are surprisingly efficient across a broad range of concentration. In similar titrations of LacI-induced looping as a function of HU concentration, HU greatly enhanced looping in 400 but not the longer 900 bp loops. Magnetic tweezers were used to investigate the effect of supercoiling on the formation of LacI-mediated DNA loops using 2 kbp DNA molecules with a centrally located O1-400-O2. Even slight tension in the magnetic tweezer interfered with looping, however, supercoiling compensated for increased tension. Positive supercoiling enhanced loop formation but less efficiently than negative which increased the looping probability from 0 to 100%. Thus the levels of accessory proteins and supercoiling, two dynamic parameters, enable modulation of regulatory loops the baseline probability of which is established by loop size and binding site affinities.

Biotechnology by Design: An Introductory Level, Project-Based, Synthetic Biology Laboratory Program for Undergraduate Students.

Synthetic biology offers an ideal opportunity to promote undergraduate laboratory courses with research-style projects, immersing students in an inquiry-based program that enhances the experience of the scientific process. We designed a semester-long, project-based laboratory curriculum using synthetic biology principles to develop a novel sensory device. Students develop subject matter knowledge of molecular genetics and practical skills relevant to molecular biology, recombinant DNA techniques, and information literacy. During the spring semesters of 2014 and 2015, the Synthetic Biology Laboratory Project was delivered to sophomore genetics courses. Using a cloning strategy based on standardized BioBrick genetic "parts," students construct a "reporter plasmid" expressing a reporter gene (GFP) controlled by a hybrid promoter regulated by the lac-repressor protein (lacI). In combination with a "sensor plasmid," the production of the reporter phenotype is inhibited in the presence of a target environmental agent, arabinose. When arabinose is absent, constitutive GFP expression makes cells glow green. But the presence of arabinose activates a second promoter (pBAD) to produce a lac-repressor protein that will inhibit GFP production. Student learning was assessed relative to five learning objectives, using a student survey administered at the beginning (pre-survey) and end (post-survey) of the course, and an additional 15 open-ended questions from five graded Progress Report assignments collected throughout the course. Students demonstrated significant learning gains (p < 0.05) for all learning outcomes. Ninety percent of students indicated that the Synthetic Biology Laboratory Project enhanced their understanding of molecular genetics. The laboratory project is highly adaptable for both introductory and advanced courses.

Exploring LacI-DNA dynamics by multiscale simulations using the SIRAH force field.

The lac repressor protein (LacI) together with its target regulatory sequence are a common model for studying DNA looping and its implications on transcriptional control in bacteria. Owing to the molecular size of this system, standard all-atom (AA) simulations are prohibitive for achieving relevant biological time scales. As an alternative, multiscale models, which combine AA descriptions at particular regions with coarse-grained (CG) representations of the remaining components, were used to address this computational challenge while preserving the relevant details of the system. In this work, we implement a new multiscale approach based on the SIRAH force field to gain deeper insights into the dynamics of the LacI-DNA system. Our methodology allows for a dual resolution treatment of the solute and solvent, explicitly representing the protein, DNA, and solvent environment without compromising the AA region. Starting from the P1 loop configuration in an undertwisted conformation, we were able to observe the transition to the more stable overtwisted state. Additionally, a detailed characterization of the conformational space sampled by the DNA loop was done. In agreement with experimental and theoretical evidence, we observed the transient formation of kinks at the loop, which were stabilized by the presence of counterions at the minor groove. We also show that the loop's intrinsic flexibility can account for reported FRET measurements and bent conformations required to bind the CAP transcription factor.

Effect of helix length on the stability of the Lac repressor antiparallel coiled coil.

The helix length dependence of the stability of antiparallel four-chain coiled coils is investigated using eight synthetic peptides (Lac21-Lac28) whose sequences are derived from the tetramerization domain of the Lac repressor protein. Previous studies using analytical ultracentrifugation sedimentation equilibrium experiments to characterize Lac21 and Lac28 justifies the use of a two state model to describe the unfolding behavior of these two peptides. Using circular dichroism spectropolarimetry as a measure of tetramer assembly, both chemical and thermal denaturation experiments were carried out to determine thermodynamic parameters. We found that the hydrophobic core residues provide the greatest impact on stability and, as a consequence, must reorganize the register of the antiparallel helices to accommodate the burial of the nonpolar amino acids. Addition of noncore residues appears to have only a minor effect on stability, and in some cases, show a slight destabilization.

Revisiting Allostery in the Lac Repressor Protein

The lac repressor protein (lac) is an allosterically regulated transcription factor which controls expression of the lac operon in bacteria. Binding of a small molecule inducer to a site 40A away from the DNA-binding domain relieves repression through what is thought to be local unfolding of the hinge helix. Despite decades of characterization, our understanding of this allosteric transition remains incomplete_mostly inferred from partial crystal structures. In principle, high-resolution solution NMR could provide detailed structural and dynamical information unobtainable by crystallography. However, due to lac's high molecular weight (70 kDa free, 85 kDa operator-bound), low solubility, and transient stability, such studies have been limited to the non-allosteric, isolated DNA-binding domain. We present, for the first time, a high-resolution solution NMR study of intact wild-type lac in all of its relevant functional states, including the poorly characterized lac-inducer-operator ternary complex. Non-uniform sampling was employed extensively in order to obtain backbone resonance assignments. Our initial results suggest that induction may not proceed simply through unfolding of the hinge helix but rather involves a more complex interplay between changes in structure and dynamics. Research supported by NIH GM102447.

Mapping Lac Repressor Interactions along DNA with Ultra-Fast Optical Tweezers

Transcription factors and DNA-binding proteins bind their specific target sequences with rates higher than allowed by 3D diffusion alone. Generally accepted models predict a combination of free 3D diffusion and 1D sliding along non-specific DNA [1]. One important issue in the field of protein-DNA interaction is the understanding of how proteins interact with non-cognate DNA sequences and how they find the sequence of interest along the DNA. We developed a system that permits to detect protein-DNA interaction with sub-ms temporal resolution and nanometer spatial precision [2]. Such precision allows us to discriminate between short events (milliseconds), which are non-specific, and long events (tens of seconds) considered to be highly specific, and to determine the precise location along the DNA where they occur. We chose a well-known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). We used a DNA molecule containing two copies of the O1 and one of the O3 operator placed at known distances and we obtained a map of the long interactions along the DNA molecule, corresponding to the position of the two O1 operators. Short interactions were instead spread along the whole DNA molecule but occurred with higher probability in correspondence of the operators and in their proximity. Dissociation of both classes of interactions was highly accelerated by an external load. Measurements performed in the presence of IPTG, a mimic of the inducer allactose, resulted in increased dissociation from the lac operators.[1] Monico, C. et al., Int. J. Mol. Sci. 14, 3961-3992 (2013).[2] Capitanio, M. et al., Nature Methods, 9, 1013-1019 (2012)