Target based regulation of the Tn7 transposase.

摘要

The bacterial transposon Tn7 is propagated by its five mobilization proteins, TnsABCDE, that collaborate to select a suitable site for Tn7 insertion. The presence of particular sequences within a target DNA can promote or discourage assembly of the TnsABCDE transposition machinery. For example, the transposition machinery is attracted to a specific site within the E. coli chromosome called attTn7, and thus Tn7 readily inserts into this site. However, once a copy of Tn7 is inserted into attTn7, DNA sequences at the ends of the transposon strongly discourage assembly of the Tn7 transposition machinery on the attTn7 site. In fact, even when a Tn7 element is positioned on the E. coli chromosome relatively far from attTn7 (≤190kb distant) the inhibitory sequences at the ends of Tn7 decrease transposition into attTn7. The decreased insertion of Tn7 into a target DNA that already contains a copy of Tn7 is known as “target immunity.”; This thesis focuses on how the Tns proteins sense and respond to the target DNA based “signals” that either promote or inhibit transposition. In chapter 2 we describe a nucleoprotein complex containing Tn7 and attTn7 target DNAs that only forms in the presence of the TnsABC+D proteins and the cofactors Mg2+ and ATP. We demonstrate that this complex is an authentic transposition intermediate. In addition, we show that interaction between TnsB, the transposase subunit that binds to the transposon ends, and TnsC, a target DNA binding protein that controls the activity of the transposase, is essential for assembly of this transposition intermediate.; In chapter 3 we identify mutants of TnsB that bypass “target immunity,” i.e. mutants that promote increased insertion of Tn7 into an attTn7 site that should be refractory to insertion due to sequences from the ends of Tn7 positioned nearby. We find these TnsB mutants bypass target immunity because they are unable to interact stably with TnsC. Taken together, our results suggest a model in which the same TnsB-TnsC interaction underlies both transposition and target immunity, and a third Tns protein, TnsA, determines whether a target site should receive an insertion or be avoided.