Faculty of Sciences Department of Molecular Biology AAnnaallyyssiiss ooff tthhee ccooooppeerraattiivvee iinntteerraaccttiioonnss bbeettwweeeenn CCooppGG ddiimmeerrss bboouunndd ttoo ssuubbssiitteess ooff iittss ooppeerraattoorr DDNNAA PhD Thesis by TANIA SAMIR RUBIO LEPE Madrid, 2013 Faculty of Sciences Department of Molecular Biology Analysis of the cooperative interactions between CopG dimers bound to subsites of its operator DNA by Tania Samir Rubio Lepe Dissertation submitted for the Degree of Doctor of Philosophy by the Universidad Autónoma de Madrid in the Molecular Biology PhD Program Supervised by Gloria del Solar Dongil, PhD Centro de Investigaciones Biológicas CSIC Madrid, 2013 The financial support for this research was granted by the Spanish Ministerio de Ciencia e Innovación (MICINN). The work was performed in the Centro de Investigaciones Biológicas (CIB) (Grants: BFU2007-63575, BFU2010-19597) and in the Center for Cooperative Research in Biosciences (CIC bioGUNE) (Grant: CTQ2009-10353/BQU). I was a recipient of a PreDoc JAE fellowship (JAEpre_07_00437) by the Consejo Superior de Investigaciones Científicas (CSIC). Front: ‘Model of CopG binding to its target DNA’. Taken and modified from (del Solar, G. et al. 2002). For my family and José Thanks to everybody who held in their minds that this thesis would be a reality. And specially thanks to José for not letting me give up…for his constant encouragement and financial support, otherwise, this book wouldn’t have been written. Contents Contents Page ABSTRACT / RESUMEN. .1 INTRODUCTION. 7 1. Plasmids. .9 1.1 Plasmid replication mechanisms. .9 1.1.1. Theta type. 9 1.1.2. Strand displacement. .10 1.1.3. Rolling-Circle-replication. 10 1.2. Control of plasmid replication. 13 2. Plasmid pMV158. 14 2.1. Control of pMV158 replication . 15 3. The RHH proteins. .16 3.1. Arc. .20 3.2. MetJ. 24 3.3. NikR. .28 4. Transcriptional repressor CopG. 32 OBJECTIVES. 37 MATERIALS AND METHODS . 41 Part I. CHEMICAL AND BIOLOGICAL MATERIAL ACQUISITION. 43 1. Bacterial strains and manipulation. 43 1.1. Culture media. .43 1.2. Transformation. .44 2. DNA preparations. .44 2.1. Plasmids. .44 2.1.1. Extraction of plasmids. 45 2.2. DNA fragments. 45 2.2.1. Obtainment of the 138-bp DNA fragment containing the copG gene. 46 2.2.2. Obtainment of the 239-bp DNA fragment containing the CopG operator. .46 2.2.3. Purification of DNA fragments. .47 2.3. Oligonucleotides. .47 2.3.1. Purification of oligonucleotides. .52 2.3.1.1. Purification of radiolabeled oligonucleotides. 53 2.3.2. ssDNA annealing. .53 2.4. DNA labeling. .60 2.4.1. 32P-labeling of oligonucleotides to be annealed. 60 2.4.2. 32P-labeling of DNA fragments. .61 3. Production of CopG proteins. 61 3.1. Heterologous expression of copG genes. 61 3.2. Protein overproduction and purification. 63 xi Part II. EXPERIMENTAL PROCEDURES . .65 1. Electrophoretic mobility shift assays. .65 1.1. Protein-DNA complex formation. .65 1.2. EMSA visualization and analysis. 65 2. Competitive EMSAs for determining relative binding affinities. .66 2.1. Relative binding affinity determination. 66 2.2. Competitive EMSAs for analyzing the role of each subsite in protein affinity. .66 2.3. Competitive EMSAs for analyzing the role of pairs of adjacent subsites in the cooperativity of protein binding. .67 2.4. Competitive EMSAs for analyzing the binding affinity of protein to the super-operators. 68 2.5. Competitive EMSAs for analyzing the binding of protein to super-operators consisting only of two subsites. 69 3. EMSAs for analyzing protein binding cooperativity . .70 3.1. Cooperativity analysis. .71 4. Determination of the equilibrium dissociation constant for binding of CopG proteins to the RSE . 72 4.1. KD calculation. .73 5. Footprinting experiments. 73 5.1. Hydroxyl radical footprinting . 74 5.1.1. OH• treatment of free DNA and protein-DNA complexes. .75 5.1.2. Recovery of OH•-treated samples from native PAA gels. .75 5.2. Dimethyl-sulfate footprinting . .76 5.2.1. DMS treatment of free DNA and protein-DNA complexes. 76 5.2.2. Recovery of DMS-treated samples. 76 5.3. Sequencing gels for the analysis of footprinting experiments. 77 6. Sequencing reactions (Maxam & Gilbert). .77 7. Analytical ultracentrifugation experiments. 78 7.1. Sedimentation equilibrium. 78 7.2. Sedimentation velocity. .79 8. Circular dichroism measurements. 79 8.1. Thermal denaturation. ..