The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a highmobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4- dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria

Análisis Bioquímico y Estructural del Factor de Transcripción Mitocondrial Humano A, TFAM, en Complejo con la Secuencia Promotora LSP

Peer Reviewe

Análisis bioquímico y estructural del factor de transcripción mitocondrial humano A, TFAM, en complejo con la secuencia promotora LSP

[spa] En esta tesis se ha conseguido la caracterización bioquímica, biofísica y estructural del factor de transcripción mitocondrial A humano, TFAM. TFAM presenta múltiples funciones en la biogénesis de la mitocondria y está implicada en transcripción, replicación, empaquetamiento del ADN mitocondrial, reparación del ADN mitocondrial.... Para tratar de hallar los mecanismos moleculares se procedió a la resolución de la estructura cristalográfica de TFAM en complejo con su secuencia diana de unión en el promotor LSP (Light Strand Promotor). La estructura de TFAM en complejo con una secuencia de 22 pb de LSP (Rubio‐Cosials, Sidow et al.) mostraba cómo TFAM impone una torsión global del ADN de 180°, con ambos dominios HMGbox introduciendo un punto de curvatura mediante la unión al surco menor del ADN. Estudios en solución mediante SAXS (Small Angle Xray Scattering) de TFAM en forma no unida permitieron asignar un alto grado de flexibilidad para la región connectora entre los dominios HMGbox y la cola C‐terminal, ambas regiones cargadas positivamente. Esta flexibilidad de la zona connectora permite un gran número de conformaciones diferentes para TFAM en su forma libre, donde los dos dominios HMGbox se encuentran con posiciones relativas diferentes. Posteriormente mediante estudios de spFRET (singleparticle FRET), se confirmó el sistema de curvatura del ADN definido por la estructura cristalográfica con la introducción de una torsión en éste en forma de U. Los estudios de SAXS y spFRET indicaban que esta unión presentaba un comportamiento ligeramente dinámico, posiblemente debido a la flexibilidad intrínseca que muestra la región connectora entre los dos dominios HMGbox en solución. Los resultados obtenidos permiten entender la multitud de funciones asignadas a TFAM y en concreto, su papel en la activación de la transcripción a partir de LSP.[eng] Transcription of human mitochondrial DNA requires transcription factor A (TFAM), also essential for DNA packaging and maintenance. Crystallographic analysis of TFAM in complex with an oligonucleotide encoding the light‐strand promoter (LSP) revealed for the first time a protein structure comprising two high‐mobility group (HMG) domains, which intercalate residues at two inverted DNA motifs and induce an overall DNA bend of ~180º stabilized by the inter‐domain linker. The U‐turn allows the TFAM C‐terminal tail, which recruits the transcription machinery, to approach the initiation site despite contacting a distant DNA sequence. We also show that structured protein regions contacting DNA in the crystal show high flexibility in solution, whereas both HMG domains have different DNA recognition capability. Our data suggest that TFAM bends LSP stepwise to create an optimal DNA conformation for transcriptional initiation, whilst facilitating DNA compaction elsewhere in the genome

U-turn DNA bending by human mitochondrial transcription factor A

Transcription factor A (TFAM) is involved in the transcription regulation, maintenance and compaction of the mitochondrial genome. Recent structural data on TFAM showed its mode of operation and clarified previous biochemical and genetic results. In solution, TFAM is highly dynamic. According to crystal structures of its complex with the cognate light-strand promoter (LSP) binding sequence, it intertwines and dramatically bends DNA, thereby allowing interactions with the transcription initiation machinery. Recent studies have shown TFAM sliding on non-specific DNA, which induces compaction by increasing DNA flexibility. Finally, the structural localization of disease-related TFAM mutations suggests functional impairment at the molecular level. © 2012 Elsevier Ltd.This study was supported by MINECO (BFU2009-07134), Generalitat de Catalunya (SGR2009-1366), and the EU (FP7-HEALTH-2010-261460, FP7-PEOPLE-2011-290246, FP7-HEALTH-2012-306029-2). ARC holds a JAE fellowship from CSIC.Peer Reviewe

Biochemical and biophysical parameters influencing macromolecular crystallization and X-ray diffraction quality

Póster presentado en el Congrés Internacional de Biologia de Catalunya "Global Questions on Advanced Biology : an international congress on interdisciplinary frontiers in biology", organizado por la Societat Catalana de Biologia, del 9 al 12 de julio de 2012 en Barcelona (España)One way to investigate and properly understand the function of a protein and its interaction with partners is to know its three-dimensional structure. Macromolecular crystallography is a tool that provides the three-dimensional structure at atomic level of a protein that has been previously crystallized. A protein crystal consists of a very large number of repeating units where each individual unit is known as the unit cell, with no internal crystalline symmetry and which contains the crystallized sample. In general, crystallization starts with the formation of nuclei of protein molecules in supersaturated chemical conditions. There are several techniques available for bringing a pure protein solution gradually to a supersaturated state, such as batch, microbatch, vapour diffusion by hanging or sitting drops, and seeding. Once obtained a protein crystal, a potential bottleneck is to obtain a wellordered crystal that will diffract X-rays strongly. Sometimes co-crystallization of a protein with a substrate may help the crystal quality, because the protein is structurally stabilized by the ligand, the crystal packing is more regular and this improves the X-ray diffraction pattern. In this case, a protein in complex with different substrates may result in different crystals that yield X-ray diffraction patterns of variable quality. We will present an example of crystal quality improvement of a protein/DNA complex in which we changed the design of the oligonucleotides harboring the DNA binding site, including the sequence, the length and the type of ends, blunt or cohesive. These changes modified the crystallization, as assessed by the macroscopic aspect of the crystals and the corresponding X-ray diffraction qualityPeer Reviewe

DNA-binding proteins analysed by SAXS

Small Angle X-ray Scattering (SAXS) of macromolecules in solution is a technique that allows lowresolution structural analysis of proteins and their complexes, including protein-DNA complexes. The number of SAXS studies on macromolecular particles has increased significantly in the last years, due to improvement of algorithms and beamtime availability in synchrotron facilities. One advantage of this technique is that is no limited by factors like particle size or flexibility; on the contrary, it is possible to assess these or other features, like the coexistence of particles of different sizes in the same preparation. The experimental SAXS curve allows to fit against it a theoretical curve calculated using a crystallographic atomic model or an electron microscopy shape, which might be modified to optimise the curve fitting. Fitting optimisation can be attempted by exploring the conformational space of the particle on the basis of the available model, using techniques like simple manual displacement of domains or more sophisticated ones like normal mode analysis, and select those models that altogether yield a curve that fits best; such a methodology can be useful in determining the degree of flexibility of domains or interdomain segments. Macromolecular complexes are reconstructed using static threedimensional models of the complexes or by docking the individual components; by playing with the proportion of non-interacting vs interacting partners, is possible to establish the dynamics of the complex. Furthermore, by introducing the conformational-space analysis algorithms within macromolecular complexes is possible to explore the conformational changes induced by ligands, substrates or interacting partners. We are going to present a SAXS analysis of three phylogenetically-related proteins whose structure is based on homology models, and in one case involving a protein/DNA complex. The strategies applied will be analysed and the results discussedPeer Reviewe

Human mitochondrial transcription factor A induces a U-turn structure in the light strand promoter

Human mitochondrial transcription factor A, TFAM, is essential for mitochondrial DNA packaging and maintenance and also has a crucial role in transcription. Crystallographic analysis of TFAM in complex with an oligonucleotide containing the mitochondrial light strand promoter (LSP) revealed two high-mobility group (HMG) protein domains that, through different DNA recognition properties, intercalate residues at two inverted DNA motifs. This induced an overall DNA bend of ~180°, stabilized by the interdomain linker. This U-turn allows the TFAM C-terminal tail, which recruits the transcription machinery, to approach the initiation site, despite contacting a distant DNA sequence. We also ascertained that structured protein regions contacting DNA in the crystal were highly flexible in solution in the absence of DNA. Our data suggest that TFAM bends LSP to create an optimal DNA arrangement for transcriptional initiation while facilitating DNA compaction elsewhere in the genome.This study was supported by the Ministerio de Ciencia e Innovación (grants BFU2006-09593 to M.S., BFU2009-07134 to M.S., BFU2008-02372 to M.C., CSD2006-00023), Generalitat de Catalunya (SGR2009-1366 to M.S., SGR2009-1309 to M.C., SGR2009-1352 to P.B.), the European Union (FP7-HEALTH-2010-261460 to M.S., FP7-BioNMR-2010-261863 to P.B.), and Instituto de Salud Carlos III-FIS-PI 10/00662. The Centro de Investigación Biomédica en Red de Enfermedades Raras is an initiative of the Instituto de Salud Carlos III. A.R.-C., J.F.S., N.J.-M. and P.F.-M. hold or held fellowships from Consejo Superior de Investigaciones Científicas, MICINN and Cusanswerk-Bischöfliche Studienförderung.Peer Reviewe

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

Human mitochondrial mTERF wraps around DNA through a left-handed superhelical tandem repeat

et al.The regulation of mitochondrial DNA (mtDNA) processes is slowly being characterized at a structural level. We present here crystal structures of human mitochondrial regulator mTERF, a transcription termination factor also implicated in replication pausing, in complex with double-stranded DNA oligonucleotides containing the tRNA Leu UUR gene sequence. mTERF comprises nine left-handed helical tandem repeats that form a left-handed superhelix, the Zurdo domain. © 2010 Nature America, Inc. All rights reserved.This study was supported by the Spanish Ministerio de Ciencia e Innovación (grants BFU2006-09593, BFU2009-07134, BIO2009-10576, BFU2008-02372, FIS-PI070045), Generalitat de Catalunya (SGR2009-1366 and SGR2009-1309) and the European Community (LSHG-2006-031220). A.R.-C. and P.F.-M. hold fellowships from CSIC and the Ministerio de Ciencia e Innovación. P.B. holds a Ramon y Cajal contract.Peer Reviewe

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compactio