
==== Front
Nucleic Acids Res
Nucleic Acids Res
nar
Nucleic Acids Research
0305-1048
1362-4962
Oxford University Press

39077947
10.1093/nar/gkae656
gkae656
AcademicSubjects/SCI00010
Synthetic Biology and Bioengineering
Engineered transcription activator-like effector dimer proteins confer DNA loop-dependent gene repression comparable to Lac repressor
Becker Nicole A Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN 55905, USA

Peters Justin P Department of Chemistry and Biochemistry, University of Northern Iowa, Cedar Falls, IA 50614, USA

Lewis Elizabeth Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN 55905, USA

Daby Camden L Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN 55905, USA

Clark Karl Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN 55905, USA
Department of Animal Science, Texas A&M University, College Station, TX 77843, USA

https://orcid.org/0000-0002-5043-6422
Maher L James III Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN 55905, USA

To whom correspondence should be addressed. Tel: +1 507 284 9041; Email: maher@mayo.edu
The first two authors should be regarded as Joint First Authors.

09 9 2024
30 7 2024
30 7 2024
52 16 999610004
16 7 2024
6 7 2024
11 6 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
2024
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Abstract

Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes.

Graphical Abstract

Graphical Abstract

Mayo Foundation 10.13039/100007048 NIH 10.13039/100000002 R35GM143949 Mayo Clinic College of Medicine and Science
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pmcIntroduction

Gene expression commences when RNA polymerase binds promoter DNA and initiates transcription yielding messenger RNA. Gene repression involves processes that prevent RNA polymerase binding at and/or transcription initiation from promoters. We and others have studied the Escherichia coli lac operon as a paradigm for understanding natural gene repression (1–7). A fundamental principle in repression of the lac promoter in the absence of small molecular inducers is binding of the bidentate repressor tetramer protein (LacI) to a promoter-proximal site (‘operator’) that precludes occupancy by RNA polymerase. The probability of repression is controlled by the local concentration of LacI protein at the proximal operator relative to RNA polymerase. Importantly, this local repressor concentration at the proximal operator reflects both contributions from free LacI molecules and from repressors bound at distal DNA sites, colliding with the proximal operator by DNA looping [(8,9); Figure 1A].

Figure 1. Principles of engineered DNA looping studied here. A. Repression is determined only by the fractional occupancy of the operator proximal to an E. coli promoter (dots indicate −35 and −10 elements) by a bidentate repressor (DNA-binding surfaces in dark grey). The repressed transcription start site is indicted by a bent arrow. The equilibrium describing total reporter gene repression (Kmax) due to repressor occupancy of operators (grey boxes) flanking the promoter reflects the total local concentration of repressor experienced at the proximal operator, i.e. the combination of contributions of free repressor collisions (KO) and collisions from repressor tethered at the distal operator by DNA looping (Ko+1max). B. TALE monomer showing central repeat domain (CRD) composed of 34-amino acid repeats with base-specific repeat variable diresidues (RVDs) indicated by base-specific colors, AcV5 tag (purple), N-terminal (green) and C-terminal (red) domains. The 15-bp DNA sequence recognized by TALE O2 is indicated above in 5′ → 3′ orientation. C-D. Covalent TALED design where following N-terminal AcV5 tag (purple), TALE 1 (grey CRD) lacks (C) or contains (D) a C-terminal domain (red) prior to a covalent linker (LSAGA or SIVAQL) upstream of TALE 2 (blue CRD). E-F. Schematic of DNA reporter constructs. Block arrow: lacZ. Bent arrow: transcription start site (TSS) downstream from indicated −10 and −35 promoter elements (black dots). Operators (grey boxes) are shown at a single proximal site (E) or in both proximal and distal locations (F) with variable center-to-center operator spacings indicated.

We and others have exploited the loop-dependence of lac repression to measure the physical resistance of double-stranded DNA to bending and twisting in vitro and in vivo through analysis of the effects of loop length on repression. Such studies also reveal the roles of DNA supercoiling and architectural DNA binding proteins in facilitation of DNA looping required for enhanced repression (6,10–15).

Artificial control of gene expression is an attractive goal for synthetic biology and therapy. Based on our understanding of DNA looping-dependent lac repression in E. coli, we have been extending these principles with the goal of artificial repression of bacterial promoters using designed repressor proteins capable of programmed sequence-specific DNA binding. For this purpose, we engineer Transcription Activator-like Effector (TALE) proteins found in Xanthomonas plant pathogen bacteria [Figure 1B; (16,17)]. Operator-specific TALEs are assembled using the known DNA base specificity code of Repeat Variable Diresidues (RVDs) within repeated 34-amino acid modules (16,17).

In our prior work, we created TALE dimers (TALEDs) whose dimerization was non-covalent and could be regulated by small molecules (18,19). This prior work proved DNA loop-dependent TALED-based gene repression in living E. coli in analogy with LacI. However, quantitation using an established thermodynamic model showed that repression was weaker than that driven by LacI, suggesting that non-covalent TALEDs dissociate into monomers under the strain of DNA bending and twisting required for loop-dependent repression.

Here, we address this by creating covalent TALEDs (Figure 1C, D). These constructs are first assayed in living E. coli cells using plasmid-based reporters containing only a proximal operator (Figure 1E) where repression is weak, and then using reporter constructs with both proximal and distal operators (Figure 1F) where repression is enhanced by increased proximal operator saturation by virtue of TALED looping from a distal operator whose distance from the proximal operator is systematically varied. This spacing variation can reveal length-dependent repression oscillation, a hallmark of DNA looping because of the expense of DNA twisting for operator spacings out of phase with the DNA helical repeat (20).

We report that, unlike the case of non-covalent TALEDs, properly optimized covalent TALEDs tightly repress target promoters, with behavior qualitatively and quantitatively comparable to LacI.

Materials and methods

Covalent TALED expression

TALEs were initially assembled by published methods (21). A combination of conventional and Gibson Assembly methods were used to assemble TALE(D) expression vectors (plasmids and sequences are detailed in Supplementary Tables S1 and S2). Briefly, TALE(D) expression plasmids are based on plasmid TALE-FKBP F36M (19) containing a pMB1 low-copy origin of replication (6). The TALE central repeat domain (CRD) was inserted into a single TALE containing plasmid as described (21). TALED receiver plasmid (pJ2805) was created using Gibson assembly (NEB) and gBlock duplexes (IDT) allowing for restriction endonuclease cloning of TALE CRDs specific for A and O2 operators. The two TALEs within each TALED were connected by amino acid linkers LSAGA or SIVAQL (22). TALE(D) expression levels were controlled by the use of five alternative promoter sequences (23). All TALE(D) expression is at an intermediate level (relative strength of 35) unless otherwise noted. TALE(D) proteins contain an N-terminal AcV5 epitope tag allowing the monitoring of expression levels by Western blotting (Figure 1B–D).

lacZ reporter constructs

DNA looping reporter constructs driving lacZ expression (Supplementary Table S3) were based on plasmids pJ2656 and pJ2783, derivatives of pJ2490 including an upstream multiple cloning site region (18), containing the lac uv5 promoter and a single proximal O2F or O2R operator, respectively (Figure 1E). Operator sequences and orientations are provided in Supplementary Figure S1. Oligonucleotide duplexes (IDT) and Gibson Assembly (NEB) were used to install the distal AF operator with variable base pair spacing upstream of the proximal operator (Figure 1F). Reporter constructs for distal AF and proximal O2F include operator center-to-center spacings of 94–108 bp and 160–194 bp. Reporter constructs for distal AF and proximal O2R include operator center-to-center spacings of 94–108 bp and 160–194 bp. Finally, reporter constructs for distal AF and proximal CF include operator center-to center spacings of 176–186 bp (Supplementary Table S3). All reporter constructs contain a p15A, low copy number origin of replication from a different compatibility group than plasmids expressing TALE DNA binding proteins to allow for co-transformation (24).

E. coli β-galactosidase reporter assay

TALE(D) protein expression and lacZ reporter plasmids were co-transformed into electrocompetent bacterial strain FW102 [araD(gptlac)5(StrR)] (25) and transformants selected on LB agar plates containing kanamycin and carbenicillin. Quantitation of DNA looping was accomplished by measurement of β-galactosidase activity in bacterial extracts as previously described (10). Activity in Miller Units (E) was calculated according to:

(1) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*} {\rm Miller}\ {\rm Units} (E) = \frac{{1000 * ({{{\rm OD}}_{420} - ( {1.75 * {{\rm OD}}_{550}})})}}{{t * v * {{\rm OD}}_{600}}} \end{eqnarray*}\end{document}

where ODx refers to optical density at wavelength x, t indicates reaction time (min), and v indicates assay culture volume (ml). Assays were performed with a plate reader. Reported data for all experiments represent the mean of extracts from at least four cultures derived from independent bacterial colonies repeated on two different days, with standard deviation indicated. Repression was quantitated in terms of total repression (RRT):

(2) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{{\rm RR}}_{\rm T} = \ {\left[ {\frac{{{E}_{ - {\rm protein}}}}{{{E}_{ + {\rm protein}}}}} \right]}_{{\rm two}\ {\rm operators}}\end{equation*}\end{document}

where E was determined in the absence or presence of TALE(D) proteins. RRT gives the total repression for any TALE(D) and reporter combination. The contribution to repression of free TALE(D) binding at the proximal operator is reported as RRF:

(3) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{{\rm RR}}_{\rm F} = \ {\left[ {\frac{{{E}_{ - {\rm protein}}}}{{{E}_{ + {\rm protein}}}}} \right]}_{{\rm single}\ {\rm operator}}\end{equation*}\end{document}

and the contribution of DNA looping (RRL) to total repression (RRT) is given by:

(4) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{{\rm RR}}_{\rm L} = \ \frac{{{{\rm RR}}_{\rm T}}}{{{{\rm RR}}_{\rm F}}}\end{equation*}\end{document}

In prior work (18), reporter expression in the absence of protein was assessed individually for each two-operator construct and was not found to display a discernable pattern nor deviate substantially from reporter expression of the proximal single-operator construct in the absence of protein. In the current work, reporter expression in the absence of protein from the proximal single-operator construct is taken as the baseline for each two-operator construct.

Thermodynamic model

The thermodynamic model of promoter repression used to fit data relating gene expression to the presence and spacing of operator sequences has been previously described (6,11,18). The model is based on the premise that promoter repression is determined by the degree of occupancy of the proximal operator at equilibrium. When no protein is bound to the proximal operator (fbound = 0), maximal activity is achieved. Any means of protein binding to the proximal operator decreases activity – binding of free protein or binding by virtue of DNA looping from the distal operator (a phase-dependent ‘specific loop’). The theoretical fbound with TALED protein as a function of DNA operator-to-operator spacing (sp) is modeled with four adjustable parameters (hr, Capp, Kmax and spoptimal) by evaluating the distribution of possible states of the proximal operator through a partition function for the system as follows:

(5) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{f}_{{\mathrm{bound}}} = \frac{{{K}_{{\mathrm{SL}}} + {K}_{\mathrm{O}}}}{{1 + {K}_{{\mathrm{SL}}} + {K}_{\mathrm{O}}}}\end{equation*}\end{document}

where each of the association equilibrium constants absorbs the (assumed) constant cellular concentration of TALED and is therefore dimensionless. The equilibrium constant for singly-bound TALED at the proximal operator (relative to the state of free proximal operator), KO, is experimentally determined from a control strain with an isolated proximal operator. The association equilibrium constant for the double-bound TALED (specific looped state) is given by:

(6) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{K}_{{\mathrm{SL}}} = \mathop \sum \limits_{i = - 96}^{96} {K}_{{\mathrm{max}}}{e}^{ - {{(sp - s{p}_{{\mathrm{optimal}}} + i \cdot hr)}}^2/\left( {2\sigma _{{\mathrm{Tw}}}^2} \right)}\end{equation*}\end{document}

with sp the actual spacing (bp) between operator centers, spoptimal is one spacing for optimal repression (of many within the range tested), hr is the DNA helical repeat, Kmax is the association equilibrium constant for the DNA looped state at spoptimal (and is maximal because operators are perfectly phased) and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\sigma$\end{document}Tw is the standard deviation of the torsion angle between operators (given thermal fluctuations):

(7) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\sigma _{\mathrm{Tw}}^{2} = sp\left(\frac{hr}{2\pi}\right)^{2}\frac{{\ell}{k}_{B}T}{{C}_{\mathrm{app}}} \end{equation*}\end{document}

where ℓ is the average bp separation (3.4 Å), kB the Boltzmann constant, T the absolute temperature, and Capp is the apparent torsional modulus for the DNA in the loop.

Data analysis and least-squares fitting

The adaptation of this model to the current analysis is explained in Supplemental Methods, where the various optimizations were performed using the SIH algorithm (26). Fit parameters give insight into the physical properties of the nucleoprotein loop.

Results

Characterization of components

Our goal is to develop optimized design guidelines for TALED-based loop-dependent gene repression in living cells. Complete lists of TALE(D) expression vectors and TALE(D) amino acid sequences used in this work are given in Supplementary Tables S1 and S2. We have demonstrated in prior work with two-operator systems (6,7) that placing a lower-affinity operator proximal to the promoter to be regulated and a higher-affinity operator in a distal position (Figure 1F) results in a larger effective contribution to repression from looping. We therefore first measured the DNA affinity of each designed TALE(D) by monitoring promoter repression in constructs with either of two single proximal target operator sequences (Figure 1E). Because each DNA sequence can be positioned on either the top or bottom strand of the proximal operator, four operator configurations were analyzed (Supplementary Figure S1), designated as O2F, O2R, AF and AR. In work by others, covalent TALEDs have been shown to function as effective ‘staple’ proteins, where each TALE domain binds a sequence on co-linear double helices, requiring TALEs 1 and 2 to adopt an antiparallel ‘C-shaped’ conformation (22). The binding of TALEDs in such ‘C’ conformations is therefore shown for each of the four operator orientations in Figure 2A, with either binding of TALE 1 (Figure 2B) or TALE 2 (Figure 2C). Protein nomenclature for TALEDs contains three parts, beginning with TALE 1 (O2 or A) without or with a C-terminal domain (c), then the linker sequence (L or S), and finally TALE 2 (always with a C-terminal domain).

Figure 2. Schematic illustrations (B, C) and data (D) for TALED proteins bound to the indicted single operator reporter constructs (A). A. Subscript F or R indicates forward or reverse orientation of the operator sequence [O2: 5′-GTGAGCGAGTAACAA-3′ or A: 5′-TCATGTTATAACGGA-3′] relative to the flanking promoter and reporter gene (Supplementary Figure S1). B. O2F operator with O2-A TALED; O2R operator with O2-A TALED; AF operator with A-O2 TALED; and AR operator with A-O2 TALED. C. O2F operator with A-O2 TALED; O2R operator with A-O2 TALED; AF operator with O2-A TALED; and AR operator with O2-A TALED. D. The indicated TALE(D) expression plasmids were assayed using reporter constructs with single proximal operators (indicated in inset) to calculate the contribution from free TALE(D) repression (RRF), which is normalized to cases with no TALE(D) expression (empty). RRF is presented on a logarithmic axis to emphasize differences.

Repression from TALE(D) binding at the proximal operator is shown in Figure 2D. Observed levels of TALE(D) repression relative to empty vector (RRF) suggest that TALE O2 (e.g. O2c) binds to its cognate operators (O2F or O2R, blue bars) with lower affinity than TALE A (e.g. Ac) binds to its cognate operators (AF or AR, grey bars).

Interestingly, binding of TALE O2 to an isolated O2F operator or TALE A to an isolated AF operator was more repressive than their binding to O2R or AR (Figure 2D, filled vs. open bars). This result suggests that repression is enhanced when the bulky N-terminus of the bound TALE projects toward the transcription start site (TSS; bent arrows in Figure 2B, C), presumably reflecting maximal steric interference with RNA polymerase. Two alternative covalent linker sequences, LSAGA or SIVAQL, between TALED domains were tested and found to have minor effects on repression (Figure 2D). LSAGA had previously been described (22) while SIVAQL was an alternative linker resulting from the employed cloning strategy. Repression was greater when TALE 2 (Figure 2C), as opposed to TALE 1 (Figure 2B), targets the proximal operator. The effect was independent of operator orientation, but most pronounced for O2F and AF. This again suggests effects of differential steric interference with RNA polymerase from tethered TALE domains near the promoter. Importantly, repression was greatly abrogated when the CTD of TALE 1 was absent (Supplementary Figure S2). Based on these results, we placed the higher-affinity AF operator in the distal position and the weaker O2F or O2R operator in the proximal position for all quantitative looping studies.

From published in vitro tethered particle motion studies (27) there is a known Gaussian concentration-dependence for maximal looping of a two-operator system, where too much protein unproductively saturates both operators with separate repressors, preventing looping, and too little repressor fails to support any looping. To define optimal TALED concentrations for looping in living E. coli, five promoter variants were used to express different TALE(D) concentrations in vivo (Supplementary Figure S3) with results shown in Supplementary Figure S4 (23). From the optimal promoter (relative strength of 35) western blot analysis detected similar expression levels of the various TALE(D) proteins (Supplementary Figure S5). This optimal promoter was selected for TALED expression in all subsequent looping studies.

While in vitro cyclization data demonstrate an optimal DNA separation of ∼500 bp for maximal looping (11), in living E. coli cells this maximum is known from classic experiments to be less than 200 bp (1,28). As in previous work (6,7) we have engineered reporter constructs with operator center-to-center spacings in two regimes of 94–108 bp and 160–194 bp (see Supplementary Table S3 for all lacZ expression DNA reporter constructs used in this work). Using constructs with an unrecognized CF operator in the proximal position (Supplementary Figure S1), we confirmed that TALE(D) binding to the distal operator had negligible effect on reporter expression, as expected (Supplementary Figure S6).

Measurement of DNA loop-dependent promoter repression by TALEDs

We systematically assayed eight TALED proteins (A-O2 or O2-A each with or without the CTD of TALE 1, and each with two different covalent linker sequences) using our two sets of reporter constructs (distal AF operator and either O2R or O2F proximal operator). Complete data appear in Supplementary Figure S7–S22 and Supplemental Data Spreadsheet. For any set of reporters, we envision two plausible TALED repression loop geometries (Supplementary Figure S23). In the case of reporter constructs with AF distal and O2R proximal operators bound by TALED O2c-S-Ac (Figure 3A), either a 180° DNA turn (Figure 3B, left) or a 360° DNA cycle (Figure 3B, right) can be proposed, depending on the properties of the TALED protein linker region and corresponding geometric constraints on overall TALED orientation (‘Z’ conformation versus ‘C’ conformation).

Figure 3. Schematic illustrations of TALED repression loops and selected TALED repression data with thermodynamic model fits as a function of operator spacing. A. Relatively high affinity AF operator (distal) and weaker O2R operator (proximal) flanking test promoter. B. DNA loops depicted as 180° turns (left) or 360° cycles (right), depending on indicated TALED conformation (‘Z’ at left vs. ‘C’ at right). It is unknown which loop conformation(s) occur in vivo. C-D. Total TALED repression (RRT) is the product of free TALED repression (RRF) and TALED repression due to looping (RRL). Data and best-fit model shown for O2c-S-Ac TALED protein. E-H. Same as A-D except depicting results for proximal O2F operator and Ac-S-O2c TALED.

Total repression (RRT) from all protein sources (Figure 3C) is the product of free TALED repression (RRF, horizontal line in Figure 3C) and repression specifically due to looping (RRL, Figure 3D). To quantitate these effects we applied our thermodynamic model of promoter repression (6,11,18). This model contains four adjustable parameters: hr is the DNA helical repeat, Capp is the apparent torsional modulus of the looped DNA, spoptimal is a center-to-center operator spacing for optimal repression, and Kmax is the equilibrium constant for DNA loop formation when operators are perfectly phased so that no DNA twisting is required for looping (Table 1). Consistent with previous work (6,11,18), the intrinsic bending stiffness of DNA observed in vitro is not a detectable energetic obstacle in vivo, presumably due to effects of supercoiling and architectural DNA binding proteins.

Table 1. Thermodynamic model fits

Looping protein	WT LacI
–IPTGa	WT LacI
 +IPTGa	WT LacI ratiob	O2c-FKBP (F36M)c	O2c-FKBP (F36M)d	O2c-S-Ace	Ac-S-O2ce	
DNA reporter	episome	episome		episome	both	plasmid	plasmid	
DNA operators	Osym-O2	Osym-O2		Osym-OinvB	O2-O2	AF-O2R	AF-O2F	
DNA spacings (bp)	63.5–90.5	63.5–90.5		82–90	160–192	94–194	94–194	
hr (bp/turn)	11.44 ± 0.74	10.73 ± 0.49			10.42 ± 0.20	11.55 ± 0.10	11.57 ± 0.32	
C app (× 10−19 erg-cm)	0.76 ± 0.42	0.64 ± 1.11			1.36 ± 0.21	0.55 ± 0.08	0.53 ± 0.10	
sp optimal (bp)	78.27 ± 0.53	78.82 ± 0.46			179.45 ± 0.38	179.00 ± 0.35	173.55 ± 1.28	
RRF = KO + 1	3.45	1.13	3.05	7	10.82	2.01	5.58	
K max	167.78 ± 60.60	2.39 ± 0.54	∼70f	38g	30	17.18 ± 0.95	112.10 ± 16.04	
K°+1max	48.6	2.1	23.0h	5.5i	2.80 ± 0.47	8.6	20.1	
K NSL	25.55 ± 31.29j	0 ± 2.06j						
K°+1NSL	7.42	0			1.55 ± 0.35			
aBecker et al. (2005). Parameters determined from fitting include a 95% confidence interval.

bOwing to residual repressor binding in the presence of IPTG, a ratio of each parameter (–IPTG/+IPTG) is needed to facilitate comparisons.

cBecker et al. (2018).

dBecker et al. (2020). All parameters are +TALED except when otherwise indicated. The normalized parameters were used for fitting. Parameters determined from fitting include a 95% confidence interval.

eCurrent work.

fDue to dephasing of the two data sets (±IPTG), this parameter serves as an estimate of Kmax and not a true maximum in the model.

gFitting was not performed, so maximal RRT is given as an estimate of Kmax.

hDue to dephasing of the two data sets (±IPTG), this parameter serves as an estimate of K°+1max and not a true maximum in the model.

iFitting was not performed, so maximal RRL is given as an estimate of K°+1max.

jPrevious model fitting made use of an additional parameter that captured non-specific contributions to DNA looping.

The observed fit value of hr (11.55 ± 0.10 bp/turn) is greater than typical in vitro values presumably due to negative supercoiling (6), and Capp (0.55 ± 0.08 × 10−19 erg-cm) is less than the value measured in vitro presumably due to additional flexibility imparted by the protein participating in the loop and the possible role of sequence-nonspecific architectural DNA binding proteins. The fit parameter Kmax (17.18 ± 0.95) captures the maximal total repression observed in the data (Figure 3C). When operators are in-phase for looping (e.g. 179.00 ± 0.35 bp), there is an 8.6-fold enhancement of repression (K°+1max) over that achieved by free TALED alone (Figure 3D). Energy costs to overcome the intrinsic twist stiffness of DNA to align operators in out-of-phase templates diminishes this optimal looping repression enhancement as expected for DNA looping, resulting in the classic pattern of repression oscillation with loop length, characterized by deeper valleys as operator spacing decreases (Figure 3D).

We further analyzed promoter repression with AF distal and O2F proximal reporters (Figure 3E) using TALED Ac-S-O2c, where we again can envision either a 180° DNA turn (Figure 3F, left) or a 360° DNA cycle (Figure 3F, right). Applying the thermodynamic model to experimental RRT (Figure 3G, with RRF given by horizontal lines) and RRL (Figure 3H), we again obtain estimates of hr (11.57 ± 0.32 bp/turn), Capp (0.53 ± 0.10 × 10−19 erg-cm) and spoptimal (173.55 ± 1.28 bp). We find that fit parameter Kmax (112.10 ± 16.04) is significantly larger when TALE 2 binds the proximal operator. Even with a larger RRF of 5.58 (i.e. KO + 1) this leads to an impressive 20.1-fold enhancement (K°+1max) over repression due to free TALED binding to an isolated proximal operator (Figure 3H).

Maximal total repression (Kmax) is always greater when the proximal operator is O2F versus O2R (Supplementary Table S4), and proximal operator identity influences basal repression in the absence of looping (KO, Figure 2D). Overall, the looping contribution to repression (K°+1max) of the various TALED constructs is very similar regardless of proximal operator identity (Supplementary Table S4).

Favorable and unfavorable TALED configurations

We find that inclusion of the ∼66-amino acid CTD of TALE 1 is crucial for efficient looping (Supplementary Figures S7-S10 versus Supplementary Figures S11–S14 and Supplementary Figures S15–S18 versus Supplementary Figures S19–S22). K°+1max remains less than 4 (1.76–3.81) for TALEDs lacking the CTD of TALE 1 and more than doubles (8.57–21.09) for TALEDs including the CTD of TALE1 (Supplementary Table S4), with one notable exception. TALED A-S-O2c exhibits intermediate behavior (Supplementary Figures S9 and S17) with K°+1max of 4.99 or 6.44 depending on proximal operator (Supplementary Table S4). When TALE 2 targets the proximal operator, the SIVAQL linker allows for better repression when the CTD of TALE 1 is absent, and this is the only circumstance observed where the selection of linker appears to significantly impact repression. However, regardless of the presence or absence of the CTD of TALE 1, repression due to looping is always enhanced (i.e. K°+1max is always greater in value) when TALE 2 targets the proximal operator (Supplementary Table S4).

The presented thermodynamic model fit curves and fit parameters represent the best global fitting of all operator center-to-center spacings in the two regimes of 94–108 bp and 160–194 bp. In most instances, the simple model applies reasonably well and uniformly across the entire loop length ranges. However, in certain cases the model fits reasonably well for only one of the two length regimes. Only one case (Supplementary Figure S7) resembles previous work (18), where the model produced satisfactory fits at repression minima but poor fits at repression maxima for operator spacings near 100 bp, a condition where weak non-covalent TALED dimerization was likely insufficient to overcome the DNA strain required for small DNA loops. In contrast, the cases of aberrant fitting patterns in the current work tend to present as either a lack of coherence in the oscillations of repression between 160–194 bp (Supplementary Figures S10, S17-S18, and S22) or both muted repression minima and maxima between 94–108 bp (Supplementary Figures S11–S12 and S14). It is possible that convolution of multiple DNA loop geometries (Supplementary Figure S23) combined with multiple TALED conformations (‘Z’ versus ‘C’) explain this lack of regular oscillatory behavior throughout both spacing regimes in these cases. In the majority of cases the thermodynamic model is adequate to fit repression data over both length regimes, indicative of favorable engineered repressor designs.

Quantitative comparison of gene repression by designed TALEDs and LacI

A key goal of our studies has been to determine if designed repression loops driven by programmable DNA binding proteins can mimic the remarkably tight lac promoter repression achieved by the natural LacI protein. Comparison of previous data (6,18,19) and our new experimental RRT (Figure 4A, RRF values given by horizontal lines) and RRL (Figure 4B) with accompanying thermodynamic model fits (Table 1) reveals that the covalent TALEDs demonstrate much superior repression relative to our prior non-covalent TALEDs. In fact, the current designs produce quantitative repression parameters comparable to LacI (Figure 4C–E). For example, K°+1max increased from 2.8 for a non-covalent TALED (Figure 4D) to 20.1 for a covalent TALED (Figure 4E), now similar to the looping enhancement observed for LacI (23.0; Figure 4C).

Figure 4. Summary of improvement in repression attributable to DNA looping with covalent TALED proteins. A-B. Total repression (RRT) is the product of free repression (RRF) and repression specifically due to looping (RRL). Ac-S-O2c TALED proteins (dark grey) repress strikingly better than prior non-covalent TALED proteins [magenta; (18)] and have repression characteristics comparable to LacI [cyan; (6)]; see Table 1 for details of the reporter expression systems, the identities of the DNA operators, and the ranges of center-to-center operator spacings. C-E. Summary of repression characteristics for LacI (C), non-covalent TALED (D), and the current covalent TALED (E) proteins.

Discussion

The natural lac operon teaches us that evolution has favored a switch where interference with RNA polymerase initiation is not achieved by requiring high repressor concentration to saturate a single operator that overlaps with the promoter. Rather, saturation of this control operator occurs at lower cellular repressor concentration because of the contribution to local repressor concentration from repressor collisions due to DNA looping from remote sites. It is evident that this general principle of biology reduces the required level of expressed regulatory proteins and increases the sensitivity of the switch (9). The apparent evolutionary superiority of this configuration makes it a fascinating synthetic biology challenge to recapitulate with entirely different repressor proteins. Thus, this work describes experiments to qualitatively and quantitatively compare DNA loop-dependent promoter repression in living E. coli cells by Lac repressor vs. designed covalent dimeric TALE (TALED) proteins. We show that properly optimized covalent TALEDs can be as effective as the LacI protein in DNA loop-dependent promoter repression.

Importance of TALE1 C-terminal domain

The profound requirement for the C-terminal region of TALE 1 is surprising given that previous TALED-based ‘staple’ proteins were designed without TALE1 C-terminal domains (22) and it is the central repeat domains (CRDs) that are thought to define TALE sequence specificity. It is known that TALEs can displace adjacent proteins in a highly polarized manner (29), suggesting a critical role of the C-terminal domain in the process of dislodging downstream competitors. However, in the current setting, we imagine the critical importance of the C-terminal domain of TALE 1 is in providing required flexibility between the two DNA-bound TALE domains.

Optimized TALED parameters

Our work suggests that several features of covalent TALEDs should be optimized for engineered gene repression. In order to maximize loop-dependent repression, a weak operator should be in the proximal position and a strong operator in the distal position, with center-to-center spacing corresponding to an integral number of DNA helical turns (where the DNA helical repeat value in E. coli is ∼11.5 bp/turn). There is some degree of spacing flexibility when defining center-to-center distance requirements for other systems, as was demonstrated here over 8 to 16 DNA helical turns. DNA looping mediated by covalently-linked modified dCas9 proteins has been demonstrated at 1400, 4700 and 11 700 bp separations in bacteria (30). Ideally, the expression level of the TALED should avoid simultaneous saturation of both operators by separate TALEDs. In the present work, this was accomplished empirically. For efficient looping, TALE 1 should contain a C-terminal domain, and the SIVAQL linker displays some subtle advantages compared to LSAGA. Maximal repression is observed in the proposed DNA/TALED loop geometries where the linker domain of the TALED is oriented toward the TSS.

We remain uncertain as to actual DNA/TALED repression loop geometries. DNA strain in a 180° turn is presumably lower than in a 360° cycle, but the energetics of ‘C’ versus ‘Z’ TALED conformations are unknown. Indeed, AlphaFold simulations of TALED structures are essentially linear, providing no insights about the flexibility of the linker or preference for ‘C’ versus ‘Z’ TALED conformations (Supplementary Figure S24). Both may occur and it is possible that some of the less coherent loop length-dependent results reflect a weighted average of more than one repression loop geometry. Furthermore, it remains possible that subtle differences between plasmid-based reporters and single copy, episome-integrated reporters may exist. However, these differences were not detected in prior work (18).

Practical value of very tight gene control

The present work also sought to test the hypothesis that limited repression characteristics of previous non-covalent TALEDs resulted from the weakness of the non-covalent dimerization interface, allowing loss of dimerization in short DNA loops under more severe strain (18,19). The present results with covalent TALEDs support this hypothesis, showing robust repression looping comparable to LacI. However, this improvement comes with the loss of inducibility. The available controls for the covalent TALEDs include adjusting the level of expression (or lack thereof), so that it is possible to saturate both operators with high protein expression. Small molecule inducers such as allolactose and IPTG destabilize LacI loops through allosteric effects on LacI-DNA interactions. Unlike our previous series of (weaker) non-covalent TALEDs that could be stabilized or de-stabilized by small molecules, the present series of strong TALEDs lack such features. It will be an interesting design challenge to engineer inducibility into covalent TALED loops. One approach would be to compete away binding of the distal TALE with an inducer TALE using TALE competition principles that have been reported (29). It is also possible to envision small molecule-based inducers that might inactivate TALEDs by non-covalent cross-linking of N and C termini to create inactive folded conformations. Ligand-inducible dCas9 variants have been used to selectively and reversibly establish chromatin loops in eukaryotic cells (31). It will be interesting to evaluate TALEDs for their ability to drive tight DNA looping in the context of eukaryotic chromatin, benefitting from enhancement of local repressor concentration to reduce required repressor expression.

Supplementary Material

gkae656_Supplemental_Files

Acknowledgements

Author contributions: N.A.B., J.P., K.C. and L.J.M. conceived and designed the experiments. N.A.B., E.L. and C.L.D. performed the experiments. N.A.B. and J.P. performed data analysis. N.A.B., J.P. and L.J.M. wrote the manuscript.

Data availability

The data underlying this article are available in the article and in its online supplementary material. Constructs are available from the authors upon request.

Supplementary data

Supplementary Data are available at NAR Online.

Funding

Mayo Foundation; NIH [R35GM143949 to L.J.M.]. Funding for open access charge: Mayo Clinic College of Medicine and Science.

Conflict of interest statement. None declared.
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