Abstract
Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process—a search state and a recognition state—facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state.
Highlights
Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood
TALE proteins are characterized by three conserved regions as shown in Fig. 1a: an N-terminal region (NTR) containing the type III translocation system required for secretion, a central repeat domain (CRD) that forms specific DNA contacts and a C-terminal region (CTR) containing nuclear localization signals and an acidic activation domain[7,8]
We studied a series of proteins derived from a parent TALE developed for the editing and correction of the human b-globin gene containing a mutation associated with sickle cell disease[37]
Summary
Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Sequence-specific recognition in the CRD is achieved by the repeat variable diresidues (RVDs), which are amino acids at positions 12 and 13 of each repeat that recognize one of the four nucleobases within the target-binding site[10,11] From this perspective, TALE proteins appear to be distinct from other DNA-binding proteins due to their ‘single repeat to single base’ recognition behaviour. Simple reprogramming of the RVDs within the CRD tandem repeats allows for the generation of specific DNA-binding proteins Using this approach, highly specific TALE–nuclease fusion proteins known as TALENs can be constructed by linking nuclease domains (typically the dimeric nuclease FokI) to custom-designed TALE proteins, thereby enabling the direct editing of target sequences in a large genome. Co-crystal structures and bulk biochemical studies of TALE proteins have revealed a wealth of information regarding specific binding, the dynamic interaction between TALEs and non-specific DNA during the search process is not yet fully understood
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