Suchergebnisse

The mitochondrial genome (mtDNA) is assembled into nucleo-protein structures termed nucleoids and maintained differently compared to nuclear DNA, the involved molecular basis remaining poorly understood. In yeast (Saccharomyces cerevisiae), mtDNA is a ∼80 kbp linear molecule and Abf2p, a double HMG-box protein, packages and maintains it. The protein binds DNA in a non-sequence-specific manner, but displays a distinct 'phased-binding' at specific DNA sequences containing poly-adenine tracts (A-tracts). We present here two crystal structures of Abf2p in complex with mtDNA-derived fragments bearing A-tracts. Each HMG-box of Abf2p induces a 90° bend in the contacted DNA, causing an overall U-turn. Together with previous data, this suggests that U-turn formation is the universal mechanism underlying mtDNA compaction induced by HMG-box proteins. Combining this structural information with mutational, biophysical and computational analyses, we reveal a unique DNA binding mechanism for Abf2p where a characteristic N-terminal flag and helix are crucial for mtDNA maintenance. Additionally, we provide the molecular basis for A-tract mediated exclusion of Abf2p binding. Due to high prevalence of A-tracts in yeast mtDNA, this has critical relevance for nucleoid architecture. Therefore, an unprecedented A-tract mediated protein positioning mechanism regulates DNA packaging proteins in the mitochondria, and in combination with DNA-bending and U-turn formation, governs mtDNA compaction. ; Ministry of Economy and Competitiveness (MINECO) [BFU2012-33516, BFU2015-70645-R to M.S; BIO2012- 32868, BFU2014-61670-EXP to M.O.; BFU2011-25697, BFU2014-60550-P to J.V.; FEDER CSIC13-4E-2065 to the Molecular imaging platform IBMB-PCB; Biomolecular and Bioinformatics Resources Platform (ISCIII PT 13/0001/0030) cofunded by the Fondo Europeo de Desarrollo Regional (FEDER) to M.O.]; Generalitat de Catalunya [SGR2009-1366, 2014-SGR-997 to M.S., SGR2009-1348, 2014 SGR-134 to M.O.]; Instituto Nacional de Bioinformatica; European Union [FP7- ´ HEALTH-2010-261460, FP7-PEOPLE-2011-290246, FP7-HEALTH-2012-306029-2 to M.S.; H2020-EINFRA2015-1-675728, H2020-EINFRA-2015-676556 to M.O.]; European Research Council [ERC-2011-ADG 20110209- 291433 to M.O.]; ITN Fellowship [FP7-PEOPLE-2011- 290246 to A.C.]; Structural Biology Unit at Molecular Biology Institute of Barcelona (IBMB) is a 'Maria de Maeztu' Unit of Excellence awarded by MINECO [MDM2014-0435]; Severo Ochoa Award of Excellence from MINECO (to IRB Barcelona); ICREA Academia Research (to M.O.). Funding for open access charge: Ministry of Economy and Competitiviness [BFU2015-70645-R]. ; Peer reviewed

© The Author(s) 2019. ; 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 compaction. ; Spanish Ministry of Science, Innovation and Universities, MINECO [BFU2012-33516, BFU2015-70645-R, RTI2018-101015-B-100 to M.S; BIO2015-64802-R, BFU2014-61670-EXP and BFU2014-52864-R to M.O., BIO2016-78006R to M.P.) Generalitat de Catalunya (2014-SGR-997 and 2017-SGR-1192 to M.S.; 2014-SGR-134 to M.O.), the Instituto de Salud Carlos III-Instituto Nacional de Bioinformática; the European Union (FP7-HEALTH-2010-261460, FP7-PEOPLE-2011-290246, FP7-HEALTH-2012-306029-2 to M.S.; H2020-EINFRA-2015-676556 to M.O.). The Biomolecular and Bioinformatics Resources Platform (ISCIII PT 13/000/0030 co-funded by the Fondo Europeo de Desarrollo Regional [FEDER]) [grants Elixir-Excelerate: 676559; BioExcel2:823830 and MuG: 676566] to M.O. P.F.-M. was awarded with FPI fellowship from MINECO, A.R.C. a JAE fellowship from Spanish Research Council (CSIC), an A. T-S. an FPU from Ministry of Education, Professional Formation (MEFP). A.T.-S. is doing his PhD thesis under the frame of the Doctorate Program Biochemistry, Molecular Biology and Biomedicine of the Autonomous University of Barcelona. The Structural Biology Unit at IBMB-CSIC is a "Maria de Maeztu" Unit of Excellence awarded by MINECO [MDM-2014-0435]. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO. M.O. is an ICREA Academia researcher. Funding for open access charge: Spanish Ministry of Innovation, Science and Universities, formerly Spanish Ministry of Economy and Competitiveness [BFU2015-70645-R].