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The dynamics of TAL effector – DNA interaction reveals optimal number of targeted nucleotides for maximum binding specificity
Author(s) -
Rinaldi Fabio Cupri,
Wilkins Katherine Elizabeth,
Bogdanove Adam Joseph
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.803.4
Subject(s) - dna , dna binding site , effector , biology , binding site , dna binding domain , genetics , computational biology , binding selectivity , dna sequencing , gene , promoter , transcription factor , biochemistry , gene expression
Transcriptional activator like effectors (TALEs) of plant pathogenic bacteria in the genus Xanthomonas have received special attention from the research community in recent years for their customizability as DNA targeting proteins. TALEs have evolved to bind DNA sequences in the promoters of host genes and upregulate expression. The DNA binding domain of TALEs is unique, comprising a series of modular repeats typically 34aa long that are essentially distinct only at positions 12 and 13, referred to as the RVD (repeat variable di‐residue). Individual base‐specific contacts by residue 13 across the repeats determine overall target specificity, with residue 12 playing a stabilizing role. Binding affinity and specificity of individual RVD‐nucleotide associations are increasingly well studied. However, there are still key questions concerning how TALEs can be designed to target DNA sequences with overall high binding affinity and specificity and how TALE target and off‐target sequences can be better predicted. Especially, the relationship between number of repeats and binding affinity has not yet been addressed quantitatively. For instance, in nature the average size of the TALE DNA binding domain is 17.5 repeats. Intuitively, one might assume that longer TALEs will have increased binding affinity and will be less likely to hit off‐target sites. We set out to test this assumption using biochemical methods to systematically study the relationship between the number of repeats of a set of designed TALEs and their respective binding affinities for the target DNA. We found that the binding affinity of TALEs in relation to their length saturates at approximately the average size of TALEs observed in nature. This suggests that increasing the number of repeats does not necessarily increase binding specificity and in fact has the opposite effect. Mathematically modeling the relationship supported this conclusion. Using the model improved the accuracy of TAL effector target prediction, providing empirical evidence in support of the model. This better understanding of the dynamics of TALE‐DNA interactions will benefit not only applications in biotechnology but also molecular characterization of the host‐pathogen interaction in the many important plant diseases caused by Xanthomonas spp . Support or Funding Information Bayer CropScience (BCS)National Science Foundation (NSF)National Institutes of Health (NIH)