Functional instability allows access to DNA in longer transcription Activator-Like effector (TALE) arrays.

Kathryn Geiger-Schuller,Jaba Mitra,Taekjip Ha,Doug Barrick

doi:10.7554/elife.38298

Kathryn Geiger-Schuller, Jaba Mitra + Show 2 more

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https://doi.org/10.7554/elife.38298

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Abstract

Transcription activator-like effectors (TALEs) bind DNA through an array of tandem 34-residue repeats. How TALE repeat domains wrap around DNA, often extending more than 1.5 helical turns, without using external energy is not well understood. Here, we examine the kinetics of DNA binding of TALE arrays with varying numbers of identical repeats. Single molecule fluorescence analysis and deterministic modeling reveal conformational heterogeneity in both the free- and DNA-bound TALE arrays. Our findings, combined with previously identified partly folded states, indicate a TALE instability that is functionally important for DNA binding. For TALEs forming less than one superhelical turn around DNA, partly folded states inhibit DNA binding. In contrast, for TALEs forming more than one turn, partly folded states facilitate DNA binding, demonstrating a mode of 'functional instability' that facilitates macromolecular assembly. Increasing repeat number slows down interconversion between the various DNA-free and DNA-bound states.

Highlights

Transcription activator-like effectors (TALEs) are bacterial proteins containing a domain of tandem DNA-binding repeats as well as a eukaryotic transcriptional activation domain (Kay et al, 2007; Römer et al, 2007)
Specificity is determined by the sequence identity at positions twelve and thirteen in each TALE repeat, which are referred to as repeat variable diresidues (RVDs) (Boch et al, 2009; Miller et al, 2015; Moscou and Bogdanove, 2009)
By measuring DNA-binding kinetics of Consensus TALEs (cTALEs) arrays that form 0.7, 1, and 1.4 superhelical turns, we probe the functional relevance of locally unfolded TALE states

Summary

Introduction

Transcription activator-like effectors (TALEs) are bacterial proteins containing a domain of tandem DNA-binding repeats as well as a eukaryotic transcriptional activation domain (Kay et al, 2007; Römer et al, 2007). The repeat domain binds double stranded DNA with a register of one repeat per base pair. Specificity is determined by the sequence identity at positions twelve and thirteen in each TALE repeat, which are referred to as repeat variable diresidues (RVDs) (Boch et al, 2009; Miller et al, 2015; Moscou and Bogdanove, 2009). This specificity code has enabled design of TALE-. TALE repeat domains wrap around DNA in a continuous superhelix of 11.5 TALE repeats per turn (Deng et al, 2012; Mak et al, 2012). Because TALEs contain on average 17.5 repeats (Boch and Bonas, 2010), most form over 1.5 full turns around

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