The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage λ. To delineate the structural basis of the NusA–λN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a λN peptide (residues 34–47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single λN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the α-helical conformation of the λN N terminus in complex with boxB RNA, residues 34–40 of λN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA–λN interaction modes is biologically significant, supporting an equimolar ratio of NusA and λN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and λN. Contrary to the RNA polymerase α subunit, λN binding does not stimulate RNA interaction of NusA. The results demonstrate that λN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.
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