突变体
核蛋白
生物
血浆蛋白结合
大肠杆菌
DNA
生物化学
化学
基因
作者
Jeanelle M. Spencer,Nancy L. Craig
标识
DOI:10.1096/fasebj.24.1_supplement.877.3
摘要
Tn7 is a transposable element isolated from E. coli that encodes five transposon‐specific proteins, Tns A, B, C, D, and E. TnsC is essential in the formation and activity of a Tn7 nucleoprotein complex comprised of TnsA,B,C, and D proteins with donor DNA as well as target DNA. The goal of this work is to identify and characterize the functional domains of TnsC. The Tn7 reaction mechanism suggests an interaction between TnsB and C. Crosslinking experiments were performed with a TnsB peptide and TnsC to stabilize and capture the complex. The data show that TnsB and TnsC participate in a direct physical interaction. Mass spectrometry of the complex will show which residues of TnsC interact with the peptide, and thereby reveal the TnsBC domain. In order to determine the remaining functional domains, Tn7 was used as a mutagen to generate a TnsC mutant library. The mutants were screened for transposition activity in a promoter capture assay. The mutable regions of TnsC were mapped and dominant negative mutants that, presumably, retain one or more essential activities of TnsC were identified. Future experiments will reveal which activity and, correspondingly, functional domain is disrupted. Experiments that identify the functional domains of TnsC will provide deeper insights into how Tn7 and other nucleoprotein complexes are controlled. This work was supported by Howard Hughes Medical Institute.
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