Abstract
Transposons are found in a wide variety of forms throughout the prokaryotic world where they actively contribute to the adaptive strategies of bacterial communities and hence, to the continuous emergence of new multiresistant pathogens. Contrasting with their biological and societal impact, only a few bacterial transposons have been the subject of detailed molecular studies. In this chapter, we propose a set of reliable biochemical methods as a primary route for studying new transposition mechanisms. These methods include (a) a straightforward approach termed "thermal shift induction" to produce the transposase in a soluble and properly folded configuration prior to its purification, (b) an adaptation of classical electrophoretic mobility shift assays (EMSA) combined to fluorescently labeled DNA substrates to determine the DNA content of different complexes assembled by the transposase, and (c) a highly sensitive "in-gel" DNA footprinting assay to further characterize these complexes at the base pair resolution level. A combination of these approaches was recently applied to decipher the molecular organization of key intermediates in the Tn3-family transposition pathway, a mechanism that has long been refractory to biochemical studies.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.