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Nanopore technology applied to the study of protein/DNA secretion

AutorValenzuela-Gómez, Fernando; Rodríguez-Larrea, David; Arechaga, Ignacio ; Cabezón, Elena
Fecha de publicación2017
CitaciónXL SEBBM Congress (2017)
ResumenAntibiotic resistance has become one of the most challenging problems in health care. Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. DNA transfer is mediated by a membrane-associated macromolecular machinery named Type IV secretion system (T4SS). DNA is transferred in complex with TrwC, a protein that cleaves the DNA at the origin of transfer and remains covalently bound to the 5' end. Given the large size of TrwC (over 100 kDa) it is unlikely that this DNA-protein complex could cross the secretion channel in a native state. Despite all the biochemical evidence supporting this, the mechanism underlying the secretion process is unknown. In this study, TrwC relaxase domain (TrwCr) (32 kDa) has been purifi ed and incubated with a 42-mer DNA substrate containing the origin of transfer sequence (oriT). After the cleavage reaction, the nucleoprotein complex consisting of TrwCr bound to the resulting 30-mer DNA substrate was purifi ed to homogeneity. Transfer across the membrane was analysed by nanopore technology by pulling DNA-TrwCr complexes through a heptameric α-haemolysin pore. The results clearly refl ect diff erent steps in a protein unfolding process through the nanopore. This is the first time that a protein of this size has been dragged through a α-haemolysin pore. However, there is still a high sequence of events that must be analysed, although the results clearly indicate that the nucleoprotein complex is easily unfolded. These novel findings make the new nanopore technology an exceptional model tool to study the diff erent transfer steps associated to a biological secretion system.
DescripciónResumen del póster presentado al XL Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Barcelona del 23 al 26 de octubre de 2017.
URIhttp://hdl.handle.net/10261/164864
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