Departamento de Biología Molecular and Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria- CSIC, 39011 Santander, Spain.
Nucleic Acids Res. 2023 Jul 21;51(13):6857-6869. doi: 10.1093/nar/gkad492.
Bacterial conjugation is the main mechanism for the dissemination of antibiotic resistance genes. A single DNA strand of the conjugative plasmid is transferred across bacterial membranes covalently bound to a large multi-domain protein, named relaxase, which must be unfolded to traverse the secretion channel. Two tyrosine residues of the relaxase (Y18 and Y26 in relaxase TrwC) play an important role in the processing of conjugative DNA. We have used nanopore technology to uncover the unfolding states that take place during translocation of the relaxase-DNA complex. We observed that the relaxase unfolding pathway depends on the tyrosine residue involved in conjugative DNA binding. Transfer of the nucleoprotein complex is faster when DNA is bound to residue Y18. This is the first time in which a protein-DNA complex that is naturally translocated through bacterial membranes has been analyzed by nanopore sensing, opening new horizons to apply this technology to study protein secretion.
细菌接合是抗生素耐药基因传播的主要机制。可移动质粒的单链 DNA 通过共价键与一种名为松弛酶的大型多结构域蛋白结合转移穿过细菌膜，松弛酶必须解折叠才能穿过分泌通道。松弛酶的两个酪氨酸残基（松弛酶 TrwC 中的 Y18 和 Y26）在接合 DNA 的加工中起着重要作用。我们使用纳米孔技术揭示了在松弛酶-DNA 复合物易位过程中发生的解折叠状态。我们观察到，松弛酶的解折叠途径取决于参与结合接合 DNA 的酪氨酸残基。当 DNA 与残基 Y18 结合时，核蛋白复合物的转移速度更快。这是首次通过纳米孔感应分析天然穿过细菌膜的蛋白-DNA 复合物，为将这项技术应用于研究蛋白分泌开辟了新的前景。
