Abstract
TALEs are sequence specific transcription activator found in phytopathogenic bacteria. Interestingly, their specificity is defined by triplet amino acid repeat sequences, which can recognize individual nucleotides in dsDNA. In crystal structures with mutants where the transcription effector domain has been deleted (TAL), we observe a fascinating structure where the TAL protein complete wraps around the dsDNA in a helical manner. There are a myriad of applications for an engineered site-specific DNA binding protein, with or without a transcription effector domain, such as genome editing and synthetic biology. Currently, however, there is no information regarding the kinetics or mechanism by which TALs bind to their respective sequences. Knowing the mechanism by which TALs find their targets and once there how long they remain bound will help in the design and application of TAL in monitoring transcription process. By engineering a SNAP-tagged TAL protein mutant, which can be fluorescently labeled with no transcriptional effector, used in co-localization of single molecules (COSMOS) experiment, we revealed that TAL, verified to be a highly specific target DNA binding protein, formed a very stable complex with target DNA. Moreover, We used this result as a springboard to incorporate fluorescent TAL with RNA polymerase (Holoenzyme) in single molecule experiments and found transcription process was blocked by TAL when TAL-DNA complex was formed. During the transcription, the RNA polymerase was stuck on the DNA with transcribed RNA fragments and cannot overpass the TAL protein.