Assembly of the N-dependent antitermination complex of phage lambda: NusA and RNA bind independently to different unfolded domains of the N protein.

The N protein of bacteriophage lambda activates expression of the delayed early genes of this phage by modifying RNA polymerase (RNAP) into a form that is resistant to termination signals. N binds to the boxB hairpin that forms in the nascent RNA transcript upon transcription of the nut regulatory element, and then interacts with RNAP by RNA looping. The binding of the N-boxB subassembly to the transcription complex is further stabilized by interaction with the Escherichia coli NusA protein. N, free in solution, exists as an unfolded protein that becomes partially structured upon binding specifically to boxB RNA. Because NusA does not assist in antitermination unless N is specifically bound to boxB, we have asked whether the structural change induced by binding to boxB affects the interaction of N with NusA. Using fluorescence spectroscopy, we have measured the affinity of N for NusA in the presence and absence of boxB RNA. We find that NusA binds to the unfolded N protein with a dissociation constant (Kd) of approximately 70 nM, and although N undergoes a significant structural change upon binding to boxB, the binding affinity of NusA for a N protein complexed with boxB is not altered. We have also shown that the boxA element of nut does not affect NusA binding to N-boxB. These results demonstrate that the interaction of N with NusA is independent of RNA binding, arguing that NusA must interact with an unfolded region of the polypeptide that remains unstructured even when N binds to boxB RNA. To further establish this point we isolated a truncated peptide containing the amino-terminal 36 residues of the N protein. Binding of boxB RNA to this peptide showed that all of the structural change in N that occurs upon binding to boxB RNA is localized within the amino-terminal 36 residues of N, therefore the C terminus of N, including the regions necessary for NusA binding and RNAP activation, remains unfolded when the full length N binds to boxB RNA. Thus it appears that N can be described as an unfolded multi-domain protein that becomes ordered in a modular fashion as it encounters its various binding partners within the N-dependent antitermination complex.