Difference between revisions of "Part:BBa K782012"

Revision as of 18:31, 24 September 2012

TALD:NLS:VP16

TALD label represents TAL efector 1295 from zebrafish experiments (Sander et al).

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.

VP16 is a 490-amino-acid protein that contains a core domain, which is enriched in acidic residues. The VP16 core contains both structured and unstructured regions. The structured region possesses a seat-like structure in which the seat surface recognizes and binds the TAATGARAT. On its own, however, VP16 does not bind to DNA well. Instead, VP16 is stabilized on the DNA by the unstructured region which interacts with Oct-1, HCF-1 and the DNA in the complex. In this complex, VP16 is able to activate transcription (Wysocka et al., 2003)

We designed TALE-based activators for specific gene activation, by fusing TAL effectors with the VP16 activator domain downstream of the minimal promoter. VP16 was placed on the C-terminal ends of the TALE DNA-binding domain (Figure 1).

Figure 1: Schematic representation of the activator construct

Characterization

HEK293T cells were cotransfected with TAL activator constructs, constituitively expressed by the CMV promoter, and mCitrine reporter plasmids, containing 10 binding sites for the designated TAL activator upstream of a minimal promoter (Figure 2). All experiments were executed in 3 biological replicates and repeated over 3 times with similar results. Tested TAL activator exhibited over 800-fold activation of the mCitrine reporter at reporter:activator ratios 1:1 (Figure 3).

Figure 2: Schematic representation of activation experiments. A: in the absence of a TAL activator, the expression of the reporter gene is repressed. B: when TAL activator is present, it binds to its respective binding site upstream of the minimal promoter and activates transcription of the reporter gene with the VP16 domain.

Figure 3: Testing activation of reporter gene transcription by addition of TAL activator

• VP16 domain was contributed by the host lab

References

Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698

Wysocka J. and Herr W (2003) The herpes simplex virus VP16-induced complex: the makings of a regulatory switch. TRENDS in Biochemical Sciences 28, 294-304

Sequence and Features