Figure 1: Identification of an optimal guanine-specific RVD. | Nature Communications

Figure 1: Identification of an optimal guanine-specific RVD.

From: Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains

Figure 1

(a) Design of the TALE RVD screening system. Each RVD-screening TALE (RVD-TALE) contains 12.5 repeats with RVDs 5 and 6 substituted with the 23 naturally occurring RVDs, and is fused to a Gaussia luciferase gene via a 2A peptide linker. The truncations used for the TALE is marked at the amino and carboxy termini with numbers of amino acids (aa) retained (top). Four different base-specific reporters with A, T, G and C substituted in the sixth and seventh nucleotides of the binding site are used to determine the base specificity of each RVD (middle). Each reporter is constructed by placing the TALE-binding site upstream of a minimal CMV promoter driving Cypridina luciferase (bottom). (b) Base preference of each natural RVD (top) is determined by measuring the levels of relative luminescence unit (RLU) for each base-specific reporter after background subtraction and normalization based on TALE protein expression level (top). We clustered RVDs according to their base preference after performing one-way ANOVA tests on each RVD. For RVDs with a single statistically significant reporter activity (P<0.05, one-way ANOVA), we plotted the reporter activity of the preferred base above the x axis, whereas the reporter activities for the non-preferred bases are shown below the x axis as negative. We clustered and ranked the RVDs without a single preferred base according to their total activity level. The abundance of each RVD in natural TALE sequences, as determined using all available Xanthomonas TALE sequences in GenBank, is plotted on a log scale (bottom). All bases in the TALE-binding site are color-coded (purple for A, red for T, orange for G and blue for C). NLS, nuclear localization signal; VP64, VP64 viral activation domain; 2A, 2A peptide linker; Gluc, Gaussia luciferase gene; minCMV, minimal CMV promoter; Cluc, Cypridina luciferase gene; polyA signal, poly-adenylation signal. All results are collected from three independent experiments in HEK 293FT cells. Error bars indicate s.e.m.; n=3.

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