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

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

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Designed architectural proteins that tune DNA looping in bacteria

Nucleic Acids Research ◽

10.1093/nar/gkab759 ◽

2021 ◽

Author(s):

David H Tse ◽

Nicole A Becker ◽

Robert T Young ◽

Wilma K Olson ◽

Justin P Peters ◽

...

Keyword(s):

Protein Binding ◽

Double Helix ◽

Dna Bending ◽

Lac Repressor ◽

Dna Looping ◽

Transcription Activator ◽

Dna Loops ◽

Bent Dna ◽

Architectural Proteins ◽

Architectural Protein

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

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Designed architectural proteins that tune DNA looping in bacteria

10.1101/2021.06.30.450240 ◽

2021 ◽

Author(s):

David H. Tse ◽

Nicole A. Becker ◽

Robert T. Young ◽

Wilma K. Olson ◽

Justin P. Peters ◽

...

Keyword(s):

Protein Binding ◽

Double Helix ◽

Dna Bending ◽

Lac Repressor ◽

Dna Looping ◽

Transcription Activator ◽

Dna Loops ◽

Bent Dna ◽

Architectural Proteins ◽

Architectural Protein

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

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Spatial organization of heterologous metabolic system in vivo based on TALE

Scientific Reports ◽

10.1038/srep26065 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 8

Author(s):

Lv-yun Zhu ◽

Xin-yuan Qiu ◽

Ling-yun Zhu ◽

Xiao-min Wu ◽

Yuan Zhang ◽

...

Keyword(s):

Spatial Organization ◽

Enzymatic Reaction ◽

Transcription Activator ◽

Metabolic System ◽

E Coli ◽

Artificial Dna ◽

Bio Engineering ◽

Indole 3 Acetic Acid ◽

Dna Scaffold

Abstract For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

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Faculty Opinions recommendation of Modeling the Lac repressor-operator assembly: the influence of DNA looping on Lac repressor conformation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1040365.489326 ◽

2006 ◽

Author(s):

Jim Maher

Keyword(s):

Lac Repressor ◽

Dna Looping

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Real sequence effects on the search dynamics of transcription factors on DNA

Scientific Reports ◽

10.1038/srep10072 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 40

Author(s):

Maximilian Bauer ◽

Emil S. Rasmussen ◽

Michael A. Lomholt ◽

Ralf Metzler

Keyword(s):

Transcription Factors ◽

Target Detection ◽

Diffusion Mechanism ◽

Coli Strain ◽

Dna Looping ◽

Real Sequence ◽

Facilitated Diffusion ◽

E Coli ◽

Sliding Motion ◽

Sequence Effects

Abstract Recent experiments show that transcription factors (TFs) indeed use the facilitated diffusion mechanism to locate their target sequences on DNA in living bacteria cells: TFs alternate between sliding motion along DNA and relocation events through the cytoplasm. From simulations and theoretical analysis we study the TF-sliding motion for a large section of the DNA-sequence of a common E. coli strain, based on the two-state TF-model with a fast-sliding search state and a recognition state enabling target detection. For the probability to detect the target before dissociating from DNA the TF-search times self-consistently depend heavily on whether or not an auxiliary operator (an accessible sequence similar to the main operator) is present in the genome section. Importantly, within our model the extent to which the interconversion rates between search and recognition states depend on the underlying nucleotide sequence is varied. A moderate dependence maximises the capability to distinguish between the main operator and similar sequences. Moreover, these auxiliary operators serve as starting points for DNA looping with the main operator, yielding a spectrum of target detection times spanning several orders of magnitude. Auxiliary operators are shown to act as funnels facilitating target detection by TFs.

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TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Acta Naturae ◽

10.32607/20758251-2014-6-3-19-40 ◽

2014 ◽

Vol 6 (3) ◽

pp. 19-40 ◽

Cited By ~ 94

Author(s):

A. A. Nemudryi ◽

K. R. Valetdinova ◽

S. P. Medvedev ◽

S. M. Zakian

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genome Engineering ◽

Disease Modeling ◽

Regulation Of Gene Expression ◽

Hereditary Disease ◽

Transcription Activator ◽

Wide Range ◽

High Standards ◽

Phenotype Formation

Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isnt enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.

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