A Bacterial Two-hybrid System Based On Transcription Activation: full version free software download2/24/2018 A bacterial two-hybrid selection system for. Together to activate transcription in our E. Based two-hybrid system can be used. Transcription activation. Analysis of mammalian peroxin interactions using a non‐transcription‐based bacterial two‐hybrid. A bacterial two‐hybrid system. This bacterial two-hybrid system is based on the finding that any sufficiently strong interaction. Promoter recognition and transcription activation in. A bacterial two-hybrid system based on. We have developed a bacterial two-hybrid system for the. Inadvertent activation/repression of transcription. We describe the use of a bacterial two-hybrid system for the study of protein-protein interactions in Escherichia coli. This system is based on transcription. Transcriptional activation of σ(54)-RNA polymerase holoenzyme (σ(54)-RNAP) in bacteria is dependent on a cis-acting DNA element (bacterial enhancer), which recruits the bacterial enhancer-binding protein to contact the holoenzyme via DNA looping. Using a constructive synthetic biology approach, we recapitulated such process of transcriptional activation by recruitment in a reconstituted cell-free system, assembled entirely from a defined number of purified components. We further engineered the bacterial enhancer-binding protein PspF to create an in vitro two-hybrid system (IVT2H), capable of carrying out gene regulation in response to expressed protein interactions. Compared with genetic systems and other in vitro methods, IVT2H not only allows detection of different types of protein interactions in just a few hours without involving cells but also provides a general correlation of the relative binding strength of the protein interaction with the IVT2H signal. Due to its reconstituted nature, IVT2H provides a biochemical assay platform with a clean and defined background. We demonstrated the proof-of-concept of using IVT2H as an alternative assay for high throughput screening of small-molecule inhibitors of protein-protein interaction. Transcriptional activation of σ 54-RNA polymerase holoenzyme (σ 54-RNAP) in bacteria is dependent on a cis-acting DNA element (bacterial enhancer), which recruits the bacterial enhancer-binding protein to contact the holoenzyme via DNA looping. Using a constructive synthetic biology approach, we recapitulated such process of transcriptional activation by recruitment in a reconstituted cell-free system, assembled entirely from a defined number of purified components. We further engineered the bacterial enhancer-binding protein PspF to create an in vitro two-hybrid system (IVT2H), capable of carrying out gene regulation in response to expressed protein interactions. Compared with genetic systems and other in vitro methods, IVT2H not only allows detection of different types of protein interactions in just a few hours without involving cells but also provides a general correlation of the relative binding strength of the protein interaction with the IVT2H signal. Due to its reconstituted nature, IVT2H provides a biochemical assay platform with a clean and defined background. We demonstrated the proof-of-concept of using IVT2H as an alternative assay for high throughput screening of small-molecule inhibitors of protein–protein interaction. Introduction Protein interactions (protein–protein, protein–nucleotide (DNA, RNA), and protein–small-molecule interactions) underlie most biological functions. However, we know far more about protein sequences than protein functions, owing largely to the rapid advances of next-generation DNA/RNA sequencing technologies. It is therefore highly desirable to develop next-generation protein technologies that allow rapid characterization of protein functions, especially protein interactions. Current approaches for protein interactions, for example, isothermal titration calorimetry and fluorescence polarization, often require costly instruments and extensive protein purification and labeling and therefore are time-consuming and limited to a few protein targets at one time. The cell-based genetic two-hybrid systems, on the other hand, have the advantages of carrying out a large number of protein interactions in each cell for selection or screening. In a typical genetic two-hybrid system, two target proteins are expressed inside the cell as hybrid proteins fused to an activation domain (AD) and a DNA(promoter)-binding domain (DB), respectively. The interaction between the target proteins recruits AD to the promoter region in the nucleus and activates the promoter-bound RNA polymerase. The issues with the genetic systems, however, are potential interferences from endogenous cellular proteins, which can lead to false negative or false positive results,, toxicity of some expressed protein interactions, and accessibility to targets due to cellular membranes and efflux pumps. Cell-free systems in general have advantages over cell-based systems for protein function studies. Without the need to grow and genetically manipulate cells, proteins (including toxic proteins) can be made and tested in a few hours in cell-free systems. Without the barrier of a cell wall or membrane, a variety of conditions, such as addition of labeled or unnatural amino acids and small-molecule inhibitors, can be applied to cell-free systems. Cell-free split-protein systems (or protein fragment complementation assays,) have been developed for in vitro protein interaction studies and have additional advantages of simultaneously expressing the target proteins and detecting their interactions via simple reporter assays. − In the absence of the protein–protein interaction, the split fragments of the reporter by themselves cannot reassemble into the active form. The interaction between two target proteins, each of which is fused to a reporter fragment, results in the reconstitution of the activity of the reporter. In this work, we intend to create a synthetic in vitro two-hybrid system (IVT2H) from a reconstituted cell-free system. We chose the two-hybrid approach because the protein interaction in a two-hybrid system only has to bring the activation domain to the vicinity of the RNA polymerase, which can result in activation of the expression of an intact reporter. In comparison, the detection of the protein interaction in a split-reporter system requires the precise alignment of the active site residues of the split reporter, and the reconstitution of its native structure while it is fused to two interacting proteins. Even under a strong protein–protein interaction, the reconstituted split reporter can have a significantly lower activity than the intact (nonfragmented) reporter, suggesting that a majority of split fragments do not form the native structure. We reason that the two-hybrid system is potentially less affected by protein conformation than the split-reporter approach. We chose the reconstituted cell-free system because it has additional advantages of lacking most cellular proteins and activities, allowing in vitro reconstruction of the process of bacterial transcription initiation in the absence of other regulatory factors., Building on our previous work, we here report the creation of the first cell-free equivalent of the genetic two-hybrid systems. Engineering Bacterial Transcription Regulation in the Reconstituted Cell-Free System The design principle of IVT2H (Figure ) was based on the process of transcriptional activation of σ 54-RNAP in Escherichia coli, due to the similarity to the eukaryotic mode of gene activation. Σ 54-RNAP forms an inactive transcriptional initiation complex on a σ 54 promoter ( pspA), which can be activated in E. Coli by the bacterial enhancer binding protein PspF., PspF functions by binding to the upstream activation sequences (UAS) near the promoter and contacting the promoter-bound σ 54-RNAP via DNA looping stabilized by the binding of integration host factor (IHF)., As a transcriptional activator, PspF is a modular protein, consisting of an N-terminal activation domain (AD), which forms a hexamer in order to activate transcription, and a C-terminal DNA binding domain (BD), which binds UAS (I and II) to facilitate the oligomerization of AD. Like the transcription factors in the genetic two-hybrid systems, PspF was the basis for constructing the hybrid fusion proteins in IVT2H (Figure and Figure S1B, ). The components of IVT2H were derived from a reconstituted bacterial transcription and translation system. In addition to the purified E. Coli translation components and T7 RNA polymerase, the protein components of IVT2H included purified E. Coli RNA polymerase core enzyme (RNAP), recombinant E. Coli IHF, and RNase inhibitor. Coli σ 54 was added as a purified protein or expressed during the IVT2H reaction from a DNA construct (Figure S1A,, P T7-σ 54). Design principles of IVT2H. (A) Detection of protein–DNA interaction. PspF or a hybrid fusion protein (AD-BD) is constitutively expressed under T7 promoter (T7) from an input DNA by T7 RNA polymerase (step 1). By binding to UAS, AD-BD is recruited. To demonstrate PspF-dependent, enhancer-specific transcription activation (Figure A), we expressed the full-length PspF (AD-BD) or its activation domain (AD) under T7 promoter in an IVT2H reaction containing a reporter DNA pspA-Fluc expressing firefly luciferase (Fluc) under the pspA promoter with UAS I and II (Figure A and Figure S1B,C, ) and measured the luciferase activities of aliquots after incubation at 37 °C. The expression of PspF resulted in a significant luciferase activity, whereas in the absence of the DNA binding domain, no significant luciferase activity was observed (Figure A, PspF and AD, gray columns). The data suggest that the binding of the expressed full-length PspF to the reporter DNA pspA-Fluc was necessary to activate the expression of Fluc in IVT2H, consistent with previous in vivo studies or in vitro experiments using purified PspF and AD., Next, we used the lambda repressor protein Cro to replace the DNA binding domain (BD) of PspF, generating a hybrid fusion protein AD-Cro. Cro binds to its consensus operator sequence ( consensus) as a homodimer., Accordingly, we replaced the PspF BD-specific UAS I and II in pspA-Fluc with 2 copies of the Cro consensus operator sequence to generate another reporter DNA 2xcons-Fluc (Figure S1C, ). The expression of AD-Cro resulted in a significantly higher luciferase activity from 2xcons-Fluc than that from pspA-Fluc (Figure A, AD-Cro, white vs gray columns), whereas the expression of PspF generated higher luciferase from the wild-type promoter in pspA-Fluc than that from 2xcons-Fluc (Figure A, PspF, gray vs white columns). The data suggest that we could fuse a different DNA binding domains to the activation domain of PspF and activate transcription simply by inserting a corresponding DNA recognition sequence upstream of the promoter. Two copies of the Cro consensus sequence in 2xcons-Fluc seemed to be sufficient to induce the hexamer formation of AD to activate transcription, presumably by recruiting AD-Cro near the promoter and increasing the local concentration of AD. Use of one copy of the Cro consensus sequence decreased the reporter expression, whereas more copies of the Cro consensus sequences did not further increase the reporter expression (Figure S2A, ). Taken together, these results not only demonstrate that we have recapitulated the PspF-mediated transcription activation in vitro but also suggest that IVT2H could potentially be used to study protein–DNA interaction (Figure A), a concept well-known as the yeast and bacterial one-hybrid systems. Protein–DNA Interaction To further demonstrate the above application, we replaced Cro with the DNA binding domain of zinc-finger protein Zif268 (Zif). Zif binds to its consensus operator sequence ( zif) as a monomer with K d = 0.5–5 nM., In comparison, Cro binds to its consensus operator sequence with a much higher affinity ( K d = 1.2 pM). We used four copies of Zif consensus operator sequence as UAS to construct a reporter DNA 4xzif-GFP, expressing a green fluorescent protein for the hybrid fusion protein AD-Zif (Figure S1C, ). To facilitate the comparison, we constructed 2xcons-GFP for AD-Cro. The expression of AD-Cro resulted in a significantly higher GFP fluorescence from 2xcons-GFP than AD-Zif from 4xzif-GFP, suggesting that the higher DNA binding affinity led to stronger transcription activation (Figure B, first and third columns). However, it has been postulated that the high affinity of Cro is due to its dimerization coupled to DNA binding,, whereas Zif binds DNA as a monomer without the cooperativity. We therefore suspected that the high fluorescence signal of AD-Cro could be the synergistic effect of Cro dimerization and DNA binding on enhancing the hexamer formation of AD and transcription activation. To address this question, we fused AD to a Cro monomeric mutant Cro K56[DGEVK]. The expression of the hybrid fusion protein AD-Cro K56[DGEVK] resulted in a drastic decrease in the fluorescent signal from 2xcons-GFP (Figure B, second column) compared with AD-Cro and to a similar level as that of AD-Zif from 4xzif-GFP (Figure B, third column). The data are consistent with previous studies suggesting that the K56[DGEVK] mutation reduced Cro DNA binding affinity by >2000-fold. Taking a step further, we added a synthetic leucine zipper (AA4) to the C-terminus of Zif to artificially homodimerize Zif. The expression of the resulting hybrid fusion protein AD-ZifAA4 indeed increased GFP fluorescence significantly compared with AD-Zif from the same reporter DNA ( 4xzif-GFP) (Figure B, fourth and third columns). As control experiments, AD-Cro K56[DGEVK] or AD-Zif was expressed in IVT2H with a reporter DNA containing a nonspecific UAS ( 4xAT-GFP, Figure S1C, ). Both hybrid fusion proteins generated significantly higher GFP fluorescence from the specific UAS ( 2xcons and 4xzif, respectively) than from the nonspecific UAS (Figure S2B,, white vs gray columns). Protein–Protein Interaction To demonstrate real-time detection of protein–protein interaction in IVT2H (Figure B), we first chose FK506 binding protein (FKBP) and FKBP-rapamycin binding domain of mTOR (FRB), known to form a heterodimer in the presence of rapamycin. The hybrid fusion proteins, AD-FKBP and FRB-Cro were coexpressed in the IVT2H reactions containing the reporter DNA 2xcons-GFP in the presence or absence of rapamycin. The fluorescent signal of each reaction was monitored in real-time during incubation at 37 °C. In the presence of rapamycin, GFP fluorescence increased significantly after ~60 min and reached a saturation after ~6 h (Figure A, left panel, +rapamycin). In the absence of rapamycin, only a small increase in the GFP fluorescence was observed over a period of 8 h (Figure A, left panel, −rapamycin). Here we successfully demonstrated the concept of the genetic two-hybrid system in IVT2H, whereby the specific interaction between FKBP and FRB from expressed hybrid fusion proteins resulted in transcription activation and GFP expression. Using FKBP and FRB, we further demonstrate the use of IVT2H as a reverse two-hybrid system in which the protein–protein interaction represses the reporter expression (Figure C). In the reverse IVT2H, the FKBP and FRB interaction was designed to activate the expression of an E. Coli transcription repressor anti-σ 28, which inhibits the GFP expression under a σ 28 promoter fliC by binding to a coexpressed σ 28 (Figure C). In addition to the DNA constructs for AD-FKBP and FRB-Cro, the reverse IVT2H reaction contained a repressor DNA ( 2xcons-anti-σ 28), a σ 28-expressing DNA (P T7-σ 28), and a reporter DNA ( flicC-GFP) (Figure S1A,B, ). In contrast to the IVT2H reaction (Figure A, left panel), the presence of rapamycin in the reverse IVT2H reaction resulted in a low GFP signal (Figure A, right panel, +rapamycin), whereas the absence of rapamycin led to a significant increase in the GFP signal (Figure A, right panel, −rapamycin). Detection of protein–protein interaction in IVT2H. (A) Binary protein–protein interaction between FKBP and FRB in IVT2H (left panel) and reverse IVT2H (right panel). AD-FKBP and FRB-Cro were expressed in the presence (+, solid line). To further establish IVT2H for detecting binary protein–protein interactions, we tested a number of other protein pairs with a wide range of known affinities (Table S1, ). We observed a remarkable correlation of the intensity of GFP fluorescence with the reported K d of the protein–protein interaction (Figure B, between 10 and 1000 nM). However, no obvious correlation was observed when K d is below 10 nM (Figure B, between 0.1 and 10 nM). The data suggest that the affinity of the binary protein–protein interaction, if between 10 nM and 1.0 μM, primarily determined the amount of the synthesized reporter protein under the IVT2H conditions. Despite the dynamic nature of the IVT2H reaction in which the concentrations of the synthesized proteins change over time and are limited by the overall protein synthesis capacity, the correlation of the protein interaction affinity with the signal output in IVT2H can potentially be described by a mathematical model for three-component binding equilibria. The formation of a ternary complex of AD-X, Y-Cro, and the reporter DNA in IVT2H is a critical step for transcription activation (Figure B) and therefore is directly correlated to the GFP expression. In the equilibria of three components, AD-X, Y-Cro, and the reporter DNA, the concentration of the ternary complex is determined by the concentration of each component, the affinity between X and Y ( K XY) and the affinity between Cro and its specific UAS ( K CroDNA) (Figure S2C, ). Cro binds its UAS with a K d of 1.2 pM, making the affinity of X and Y a limiting factor for the ternary complex formation. Using simulations provided by the mathematical model, we plotted the dose–response curves of the ternary complex at different affinities of X and Y (Figure S2D, ). The results (see the legend in Figure S2D, ) are consistent with not only the observed correlation between the GFP expression and the affinity of the protein–protein interaction (between 10 and 1000 nM, Figure B) but also with the observed noncorrelation at affinities below 10 nM (between 0.1 and 10 nM, Figure B). Protein–RNA Interaction We further demonstrated the use of IVT2H as a three-hybrid system for the detection of protein–RNA interactions (Figure D). As the hybrid fusion proteins, we chose the first 22 residues of the N-protein of the bacteriophage lambda (λN22) and the coat protein of the Pseudomonas phage PP7 (PP7CP), generating AD-λN22 and PP7CP-Cro, respectively. Since λN22 and PP7CP bind to their hairpin RNA substrates, λboxB and PP7, respectively, we created a substrate RNA construct (P T7 λboxB-PP7), which can produce a hybrid RNA substrate (λboxB-PP7) from T7 promoter during the IVT2H reaction. As a control, PP7 was replaced by TAR, a hairpin RNA from HIV, to generate a nonsubstrate construct (P T7 λboxB-TAR). In the IVT2H reactions expressing both AD-λN22 and PP7CP-Cro, addition of P T7 λboxB-PP7 resulted in significant luciferase activity (Figure, third column), whereas in the absence of P T7 λboxB-PP7 or the presence of the nonsubstrate RNA (P T7 λboxB-TAR), no significant luciferase activity was observed (Figure, first and second columns, respectively). Expression of only one hybrid protein (AD-λN22) also failed to generate significant luciferase activity (Figure, fourth column). The data suggest that the specific binding of the hybrid fusion proteins to both RNA substrates activated the expression of the luciferase reporter. Use of IVT2H as an in vitro assay for qHTS of small-molecule inhibitors of protein–protein interaction. (A) qHTS three-axis plot of the activity of 67-member steroidal library (listed in Supplementary Table 2, ) against E2-mediated. In summary, we recreated the process of bacterial enhancer-specific gene activation in a reconstituted cell-free system using a bottom-up synthetic biology approach. We further engineered such synthetic system to create IVT2H and demonstrated its broad utility as a universal assay format for protein interactions. Unlike cell-based genetic systems that require manipulating cellular genetic backgrounds for detecting different types of protein interactions, IVT2H can be formulated into one-hybrid, two-hybrid, reverse two-hybrid, or three-hybrid assay simply by changing DNA constructs, while the protein components of IVT2H remain unchanged. Such flexibility of IVT2H can be harnessed for more applications. For instance, IVT2H can be constructed as a reverse two-hybrid assay for screening small-molecule inhibitors, allowing inhibition to generate a positive signal (Figure S3B, ). As a three-hybrid system, IVT2H can potentially be used to detect not just RNA but any molecule in solution (Figure D). Detection of protein interactions in IVT2H requires the same reconstituted protein translation machinery to synthesize multiple proteins (interacting proteins, σ 54, and reporter protein) in coupled steps. In in vitro reactions, IVT2H has a defined capacity for protein synthesis; therefore, the amounts of DNA in IVT2H affect the optimal signal-to-noise ratio. Since the interacting proteins and σ 54 are expressed under the strong T7 promoter and the reporter under the relatively weak σ 54 promoter, the DNA concentrations in IVT2H are adjusted to the picomolar range for the interacting proteins and σ 54 and the nanomolar range for the reporter. By varying the DNA concentrations, we determined that the optimal DNA concentration for the DNA-interacting protein is ~10 pM and that for protein–protein interacting pairs is ~50 pM (Figure S4A,B, ). Higher DNA concentrations resulted in higher amounts of the interacting proteins (Figure S5A,B, ) but lower GFP reporter signals (Figure S4A,B, ), likely due to the limited synthesis capacity of IVT2H. The optimal DNA concentrations and the correlations of the DNA concentrations with the protein amounts in IVT2H have been carefully characterized for only a few proteins (Figure S5, ). It is possible that some interacting proteins are not expressed or are expressed at unusually high levels, which could lead to false negative or false positive signals in IVT2H. At least 4.4–10 nM of the reporter DNA is needed in IVT2H to generate sufficient GFP signal (Figure S4C, ). Further increasing the reporter DNA concentrations can result in a stronger GFP signal but also a higher signal from the nonspecific interaction (Figure S4C, ). In addition, using higher reporter DNA concentrations increases the cost of making DNA. The protein components of IVT2H can be readily purified or obtained from commercial sources (Table S4, ). Cell-free split-protein systems represent a similar approach to IVT2H for in vitro detection of a variety of protein interactions., Like IVT2H, split-protein systems can also be applied for screening small-molecule inhibitors of protein–protein interaction. The signal from the cell-free split-protein assay is dependent on the reconstitution of the activity of the reporter protein, potentially can be monitored in real-time, but is not coupled to a gene activation event (at least in vitro). In IVT2H, protein interaction is coupled to the activation of a reporter gene via domain recruitment. Consequently the signal is amplified by the transcription and translation of the nonfragmented reporter protein. In addition, we show that the signal from IVT2H is correlated to the relative strength of the protein interaction. Conclusions Though a variety of genetic systems and in vitro systems for detecting protein interactions have been developed, direct comparison of these methods against the same reference sets reveals that each method has inherent limitations and often detects a subset of interactions. In this work, we develop IVT2H as a unique cell-free system for detection of protein interactions, which is clearly distinct from cell-based methods and cell-free split-protein systems. IVT2H may serve as an alternative and independent tool for validating existing protein interaction data and providing new ones. Without using cells, IVT2H may detect “elusive” protein interactions, such as those of difficult-to-express proteins or those toxic to cells. The next step for IVT2H is its integration with high-throughput platforms to allow selection or screening of protein interactions, a current advantage of cell-based genetic systems. Cell-free systems are compatible with liposomes and microdroplets; thus use of IVT2H for high throughput protein interaction studies is highly possible. As a reconstituted synthetic biological system, IVT2H contains a defined number of components with known initial concentrations, thus representing a unique experimental model for computational simulations of synthetic gene circuits or chemical biology of gene regulation. Reconstitution of IVT2H IVT2H was based on previous work that coupled E. Coli transcriptional machinery to the reconstituted protein synthesis system., IVT2H typically contained the reconstituted protein synthesis system (with T7 RNA polymerase),, purified E. Coli RNA polymerase core enzyme (New England Biolabs), purified recombinant E. Coli IHF, murine RNase inhibitor (New England Biolabs), and DNA constructs (plasmids and linear DNA) expressing sigma factors, hybrid fusion proteins, RNA substrates, and reporters (see below). DNA Constructions for IVT2H The gene for E. Coli sigma 54 (σ 54) was amplified by PCR from E. Coli genomic DNA and cloned into an expression vector pCOAT containing a T7 promoter to generate the plasmid DNA P T7-σ 54 (Figure S1A, ). The σ 54 protein was expressed in E. Coli from P T7-σ 54 as a recombinant protein with a N-terminal 6xhis tag and purified according a previous protocol. The gene for E. Coli phage-shock protein F (PspF) was amplified from E. Coli genomic DNA and cloned into pCOAT to give P T7-PspF (Figure S1B, ). Similarly, the activation domain (AD, residues 1–296) of PspF was cloned into pCOAT to give P T7-AD. The gene for the lambda repressor protein Cro was amplified from lambda phage DNA, whereas the genes for Cro K56[DGEVK], the zinc finger DNA binding domain of Zif268 (Zif), and the fusion protein ZifAA4 were synthesized commercially (Integrated DNA Technologies) (Figure S1B, ).
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