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The effect of increasing numbers of repeats on TAL effector DNA binding specificity.

ABSTRACT: Transcription activator-like effectors (TALEs) recognize their DNA targets via tandem repeats, each specifying a single nucleotide base in a one-to-one sequential arrangement. Due to this modularity and their ability to bind long DNA sequences with high specificity, TALEs have been used in many applications. Contributions of individual repeat-nucleotide associations to affinity and specificity have been characterized. Here, using in vitro binding assays, we examined the relationship between the number of repeats in a TALE and its affinity, for both target and non-target DNA. Each additional repeat provides extra binding energy for the target DNA, with the gain decaying exponentially such that binding energy saturates. Affinity for non-target DNA also increases non-linearly with the number of repeats, but with a slower decay of gain. The difference between the effect of length on affinity for target versus non-target DNA manifests in specificity increasing then diminishing with increasing TALE length, peaking between 15 and 19 repeats. Modeling across different hypothetical saturation levels and rates of gain decay, reflecting different repeat compositions, yielded a similar range of specificity optima. This range encompasses the mean and median length of native TALEs, suggesting that these proteins as a group have evolved for maximum specificity.

SUBMITTER: Rinaldi FC

PROVIDER: S-EPMC5499867 | biostudies-other | 2017 Jun

REPOSITORIES: biostudies-other

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The effect of increasing numbers of repeats on TAL effector DNA binding specificity.

Rinaldi Fabio C FC Doyle Lindsey A LA Stoddard Barry L BL Bogdanove Adam J AJ

Nucleic acids research 20170601 11

Transcription activator-like effectors (TALEs) recognize their DNA targets via tandem repeats, each specifying a single nucleotide base in a one-to-one sequential arrangement. Due to this modularity and their ability to bind long DNA sequences with high specificity, TALEs have been used in many applications. Contributions of individual repeat-nucleotide associations to affinity and specificity have been characterized. Here, using in vitro binding assays, we examined the relationship between the ...[more]

PMID: 28460076

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Project description:Delivered into plant cells by type III secretion from pathogenic Xanthomonas species, TAL (transcription activator-like) effectors are nuclear-localized, DNA-binding proteins that directly activate specific host genes. Targets include genes important for disease, genes that confer resistance, and genes inconsequential to the host-pathogen interaction. TAL effector specificity is encoded by polymorphic repeats of 33-35 amino acids that interact one-to-one with nucleotides in the recognition site. Activity depends also on N-terminal sequences important for DNA binding and C-terminal nuclear localization signals (NLS) and an acidic activation domain (AD). Coding sequences missing much of the N- and C-terminal regions due to conserved, in-frame deletions are present and annotated as pseudogenes in sequenced strains of Xanthomonas oryzae pv. oryzicola (Xoc) and pv. oryzae (Xoo), which cause bacterial leaf streak and bacterial blight of rice, respectively. Here we provide evidence that these sequences encode proteins we call "truncTALEs," for "truncated TAL effectors." We show that truncTALE Tal2h of Xoc strain BLS256, and by correlation truncTALEs in other strains, specifically suppress resistance mediated by the Xo1 locus recently described in the heirloom rice variety Carolina Gold. Xo1-mediated resistance is triggered by different TAL effectors from diverse X. oryzae strains, irrespective of their DNA binding specificity, and does not require the AD. This implies a direct protein-protein rather than protein-DNA interaction. Similarly, truncTALEs exhibit diverse predicted DNA recognition specificities. And, in vitro, Tal2h did not bind any of several potential recognition sites. Further, a single candidate NLS sequence in Tal2h was dispensable for resistance suppression. Many truncTALEs have one 28 aa repeat, a length not observed previously. Tested in an engineered TAL effector, this repeat required a single base pair deletion in the DNA, suggesting that it or a neighbor disengages. The presence of the 28 aa repeat, however, was not required for resistance suppression. TruncTALEs expand the paradigm for TAL effector-mediated effects on plants. We propose that Tal2h and other truncTALEs act as dominant negative ligands for an immune receptor encoded by the Xo1 locus, likely a nucleotide binding, leucine-rich repeat protein. Understanding truncTALE function and distribution will inform strategies for disease control.

Dataset Information

The effect of increasing numbers of repeats on TAL effector DNA binding specificity.

Publications

The effect of increasing numbers of repeats on TAL effector DNA binding specificity.

Similar Datasets

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