Protein flexibility and synergy of HMG domains underlie U-turn bending of DNA by TFAM in solution

Human mitochondrial transcription factor A (TFAM) distorts DNA into a U-turn, as shown by crystallographic studies. The relevance of this U-turn is associated with transcription initiation at the mitochondrial light strand promoter (LSP). However, it has not been yet discerned whether a tight U-turn or an alternative conformation, such as a V-shape, is formed in solution. Here, single-molecule FRET experiments on freely diffusing TFAM/LSP complexes containing different DNA lengths show that a DNA U-turn is induced by progressive and cooperative binding of the two TFAM HMG-box domains and the linker between them. SAXS studies further show compaction of the protein upon complex formation. Finally, molecular dynamics simulations reveal that TFAM/LSP complexes are dynamic entities, and the HMG boxes induce the U-turn against the tendency of the DNA to adopt a straighter conformation. This tension is resolved by reversible unfolding of the linker, which is a singular mechanism that allows a flexible protein to stabilize a tight bending of DNA.This work was supported by the Ministry of Economy and Competitiveness (MINECO) (BFU2012-33516 and BFU2015-70645-R to M.S., and BIO2012-32868 and BFU2014-61670-EXP to M.O.); Generalitat de Catalunya (SGR2009-1366 and 2014-SGR-997 to M.S., and SGR2009- 1348, 2014 SGR-134 to M.O.); the Instituto Nacional de Bioinforma´tica; the European Union (FP7-HEALTH-2010-261460, FP7-PEOPLE-2011- 290246, and FP7-HEALTH-2012-306029-2 to M.S., and H2020-EINFRA-2015-1-675728 and H2020-EINFRA-2015-676556 to M.O.); and the European Research Council (ERC-2011-ADG_20110209-291433 to M.O.). A.R.-C. was awarded with a ‘‘Junta para la Ampliacio´n de Estudios’’ (Programa JAE) fellowship from Consejo Superior de Investigaciones Cientı´ficas (CSIC). The Structural Biology Unit at IBMB-CSIC is a ‘‘Maria de Maeztu’’ Unit of Excellence awarded by the Ministry of Economy and Competitiveness (MINECO) under MDM-2014-0435. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Ministry of Economy and Competitiveness (MINECO). The CBS is a member of the French Infrastructure for Integrated Structural Biology (FRISBI), a national infrastructure supported by the French National Research Agency (ANR-10-INBS-05).Peer reviewe