Local evolutionary landscapes of λ N-boxB and HK022 Nun-boxB Interactions.

Abstract

Expression of λ delayed-early genes requires overcoming transcription terminators through an antitermination process initiated by an arginine-rich motif (ARM) binding a regulatory RNA in a λ N ARM-boxB interaction. HK022 Nun protein competes with N to bind boxB and induces premature λ transcription termination. Although structural data support very similar N ARM-boxB and Nun ARM-boxB interactions, recently published mutagenesis data suggests Nun-NusG contacts through non-conserved but required Asp26, Trp33, and Arg36 residues embedded in a functionally important exposed hydrophobic ridge. This prompted a further examination of the interactions to elucidate their differences. In this study, mutational analyses and specificity assessments of boxBs with expanded and unexpanded loops shows Nun recruits boxB variants that N cannot, affirming that their binding modes are distinct. Scanning mutagenesis of N ARM revealed N does not have a restriction on Thr5, Ala12, and Gln15 at positions equivalent to the restriction on Asp26, Trp33, and Arg36 of Nun. Complex ARM hybrids having a specific combination of N and Nun residues characterized Nun’s recognition strategy as complex, involving a combination of multiple requirements, and likely not modular. In contrast, N’s recognition strategy is simple, more robust (resistant to changes in its amino acid sequence), and with no equivalent dependence on NusG-CTD contacts. The results elucidate the difference between N and Nun recognition strategies, allow speculations on NusG localization within the termination and the antitermination complexes, and address the possible genetic drift of N and Nun binding modes, as well as boxB sequence, along neutral networks.