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NicTAL12:NLS DNA binding domain

Introduction

TAL effectors (TALEs) are bacterial plant pathogen transcription factors, that bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. A tandem TALE repeat contains 33 to 35 amino acids, where the 12th and 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).

Figure 1: Schematic representation of the construct.

Single binding sequence for NicTAL:TCTATCAATGATAGA

Characterization

We discovered that NicTAL part deposited in the Registry by the Slovenian iGEM2010 team was missing subdomain in DNA-binding domain, so our team added this missing part on N terminal sequence. Apart from adding N terminal sequence,we added HIS tag on N terminal end and NLS on terminal C end of NicTAL12 (Figure 2). This construct was later used for designing TAL-based activator and repressor by adding VP16 and KRAB domain.

Figure 2:Sequence alignment of NicTAL10 and NicTAL12 showing differences between constructs.

Results obtained by testing NicTAL12:KRAB repressor and activator, proved that NicTAL12 binds to its binding sites better than NicTAL10.

Figure 3: Results of testing NicTAL:KRAB repressor shows, that NicTAL12 binds to DNA better than NicTAL10.

References

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.

Sequence and Features