Rationally designed coiled-coil DNA looping peptides control DNA topology

Gowetski, Daniel B.; Kodis, Erin J.; Kahn, Jason D.

doi:10.1093/nar/gkt553

Cited by 6 publications

(6 citation statements)

References 71 publications

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“…Gowetski et al. engineered DNA loops through designed coiled-coil peptides ( 32 ). Praetorius and Dietz have recently shown in vitro assembly of complex duplex DNA shapes by DNA looping anchored by designed constitutive dimeric TALE proteins ( 35 ).…”

Section: Discussionmentioning

confidence: 99%

Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins

Becker

Schwab

Clark

et al. 2018

Nucleic Acids Research

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Genetic switches must alternate between states whose probabilities are dependent on regulatory signals. Classical examples of transcriptional control in bacteria depend on repressive DNA loops anchored by proteins whose structures are sensitive to small molecule inducers or co-repressors. We are interested in exploiting these natural principles to engineer artificial switches for transcriptional control of bacterial genes. Here, we implement designed homodimeric DNA looping proteins (‘Transcription Activator-Like Effector Dimers’; TALEDs) for this purpose in living bacteria. Using well-studied FKBP dimerization domains, we build switches that mimic regulatory characteristics of classical Escherichia coli lactose, galactose and tryptophan operon promoters, including induction or co-repression by small molecules. Engineered DNA looping using TALEDs is thus a new approach to tuning gene expression in bacteria. Similar principles may also be applicable for gene control in eukaryotes.

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Section: Discussionmentioning

confidence: 99%

Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins

Becker

Schwab

Clark

et al. 2018

Nucleic Acids Research

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show abstract

Section: Discussionmentioning

confidence: 70%

“…Finally, we speculate that our results may aid in the design of DNA nanostructures (48,49). Synthetic looping proteins have been used previously to form specific DNA contacts (50) which have aided in the assembly of DNA nanostructures (14). Here, we speculate that multivalent cations with nonspecific contacts could also be used to bend, condense, or stabilize engineered DNA constructs.…”

Section: Discussionmentioning

confidence: 99%

DNA looping by protamine follows a nonuniform spatial distribution

McMillan

Kuntz

Devenica

et al. 2021

Preprint

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DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes (SMC) proteins such as condensin. Here, however, we are interested in a different looping method whereby multivalent cations (charge≥+3), such as protamine proteins, neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤ 1 μm) DNA fragments using atomic force microscopy (AFM). We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it overestimated the number of loops that form at the ends of the molecule and failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine’s in vivo role for looping DNA into toroids and the mechanism of DNA condensation by multivalent cations more broadly.SIGNIFICANCEDNA looping is important in a variety of both in vivo functions (e.g. gene regulation) and in vitro applications (e.g. DNA origami). Here, we sought a mechanistic understanding of DNA looping by multivalent cations (≥+3), which condense DNA into loops and toroids. One such multivalent cation is the protein protamine, which condenses DNA in sperm. We investigated the mechanism for loop formation by protamine and found that the experimental data was consistent with an electrostatic multibinding model in which two protamines bind electrostatically to the DNA within a 50-nm region to form a loop. This model is likely general to all multivalent cations and may be helpful in applications involving toroid formation or DNA nanoengineering.

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“…Note that in general repressor molecules could bind to operator sites of two different gene copies at the same time, which has been observed in several experiments in vitro [52, 53]. It would be very interesting to study the effect of this situation on transcriptional regulation, especially in cases where the gene is located on mobile DNA elements such as plasmids.…”

Section: Repression Architecturesmentioning

confidence: 94%

Self-consistent theory of transcriptional control in complex regulatory architectures

et al. 2017

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Individual regulatory proteins are typically charged with the simultaneous regulation of a battery of different genes. As a result, when one of these proteins is limiting, competitive effects have a significant impact on the transcriptional response of the regulated genes. Here we present a general framework for the analysis of any generic regulatory architecture that accounts for the competitive effects of the regulatory environment by isolating these effects into an effective concentration parameter. These predictions are formulated using the grand-canonical ensemble of statistical mechanics and the fold-change in gene expression is predicted as a function of the number of transcription factors, the strength of interactions between the transcription factors and their DNA binding sites, and the effective concentration of the transcription factor. The effective concentration is set by the transcription factor interactions with competing binding sites within the cell and is determined self-consistently. Using this approach, we analyze regulatory architectures in the grand-canonical ensemble ranging from simple repression and simple activation to scenarios that include repression mediated by DNA looping of distal regulatory sites. It is demonstrated that all the canonical expressions previously derived in the case of an isolated, non-competing gene, can be generalised by a simple substitution to their grand canonical counterpart, which allows for simple intuitive incorporation of the influence of multiple competing transcription factor binding sites. As an example of the strength of this approach, we build on these results to present an analytical description of transcriptional regulation of the lac operon.

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Rationally designed coiled-coil DNA looping peptides control DNA topology

Cited by 6 publications

References 71 publications

Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins

Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins

DNA looping by protamine follows a nonuniform spatial distribution

Self-consistent theory of transcriptional control in complex regulatory architectures

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