Analysis of the cooperative interactions between CopG dimers bound to subsites of its operator DNA
AdvisorSolar Dongil, Gloria del
EntityUAM. Departamento de Biología Molecular
SubjectsGenética bacteriana - Plasmidos; Biología y Biomedicina / Biología
NoteTesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-11-2013
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Protein CopG (45 aa) encoded by plasmid pMV158 is the smallest transcriptional repressor characterized so far, and is the prototype of a family of plasmids replicating by the Rolling-Circle mechanism. CopG represses Pcr promoter, which directs the transcription of its own gen (copG) and that of the initiator protein for plasmid replication (repB). The DNA target of CopG has a symmetric element (SE) that includes a repeat of the sequence 5’-TGCA-3’ at each side of the symmetry axis, in the subsites called the Left and Right Symmetric Elements (LSE and RSE, respectively). The LSE overlaps the -35 promoter region. Crystallographic studies of CopG free and bound to the SE demonstrated that it has a “β-Ribbon-α1-Helix-α2-Helix” (RHH) threedimensional configuration, and that one dimer of protein binds to each half of the SE. The CopGDNA interaction relies on the contacts made by the β-Ribbon with DNA bases, and on those made by the α2 helix N-terminal residues with the phosphate backbone. Binding of CopG to its target DNA generates, with a pattern of cooperativity, four well-defined complexes (CI, CII, CIII and CIV), which are supposed to correspond to 1, 2, 3 and 4 dimers bound to the operator DNA. Thus, one dimer binds to each subsite of the SE, and two extra dimers would bind laterally to the SE, in regions located one helical turn apart, defined as the Left and Right Arms (LA and RA, respectively). The RA overlaps the extended -10 region of Pcr promoter. In this Thesis we demonstrated that, in fact, the specific nucleoprotein complex formed by CopG consists of four dimers bound in a sequential and highly cooperative manner to four subsites of the DNA spanning about 50 bp. We also determined that the RSE is the primary site of protein binding, to which CopG binds establishing interactions beyond those described in the SE-CopG tetramer co-crystals. Then, the second dimer binds to the RA, the third to the LSE and the last one binds to the LA. These results support the hypothesis about the CopG-mediated transcriptional repression by active dissociation, according to which the RSE keeps accessible for CopG binding once the RNAP is bound to the Pcr promoter. Binding of CopG to the RSE then promotes, by cooperative interactions, the formation of the complete nucleoprotein complex, dislodging finally the RNAP. On the other hand, two CopG variants were studied: CopGA30E and CopGG25E. The altered residues in these mutants are located in the dimer-dimer interaction surface observed in the SE-CopG tetramer co-crystals. By NMR, differences in the structural configuration of the three CopG variants were not observed. However, EMSAs revealed a diminished affinity for DNA and altered cooperative properties in both mutants compared to the wild-type protein. Using the three CopG variants and a battery of operator mutants, we observed and proposed that: i) the recognition sequence that better promotes binding of CopG was the 6-bp motif 5’-(R)TGCA(Y)-3’, and ii) upon binding, the four dimers arrange as two tetramers, each bound to a half of the operator, hence generating at least two different dimerdimer interfaces in the whole nucleoprotein complex formed by CopG, namely the interaction surface observed for the two dimers bound to the SE as described in the co-crystals, and the interface generated between the two dimers bound to either of the operator halves. All together, we demonstrated that binding of CopG to its operator DNA depends both on the nucleotide sequence and on the cooperative interdimeric interactions.
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Google Scholar:Rubio Lepe, Tania Samir
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