Interaction of the intrinsically unstructured phage λ N protein with Escherichia coli NusA

N protein of the Escherichia coli phage lambda (lambda N) is involved in anti termination, a transcription regulatory process that is essential for the expression of delayed early genes during phage lytic development. lambda N is an Intrinsically unstructured protein that possesses three distinct binding sites interacting with the carboxy terminus of the E. coli host factor protein NusA, the viral nutBoxB-RNA, and RNA polymerase, respectively. Heteronuclear NMR experiments with lambda N(1-53) in complex with NusA(339-495) revealed that upon complex formation the lambda N-binding interface, lambda N(34-47), adopts a rigid structure. NMR data also indicate the induction of a weak helical structure in the nutboxB RNA-binding region lambda N(1-22) upon binding to NusA(339-495) even in the absence of RNA. Titration experiments of the lambda N(1-53)-nutBoxB RNA complex with NusA(339-495) revealed that the ternary complex can be described in terms of two structurally independent binary interactions. Furthermore, chemical-shift perturbation experiments with different NusA constructs and different lambda N peptides showed that only NusA(353-416) is involved in lambda N binding. We found that only one molecule of NusA(339-426) binds to one molecule of lambda N(1-53). We also clarified the role of the lambda N-binding region and could show that N41-R47 also binds to NusA(339-495). Furthermore, we observe that lambda N(1-22) adopts a helical fold upon binding to NusA(339-495), in agreement with one of the theoretical models of lambda N action.