Dual Role of boxB RNA Motif in the Mechanisms of Termination/Antitermination at the Lambda tR1 Terminator Revealed in vivo

Rho-dependent transcription termination at the phage lambda tR1 terminator is governed primarily by the upstream rut element that encodes two RNA regions rutA and rutB. The two regions are separated by the boxB RNA motif, which is believed to be dispensable for Rho activity but serves as a binding site for λ N protein in the antitermination process. By using a minimal in vivo termination system, we show that the intervening boxB RNA motif has a double function in the mechanisms of termination/antitermination at λtR1. As a folded hairpin structure, it acts as a clamp that holds rutA and rutB side by side for optimal interactions with Rho leading to efficient termination. Conversely, the binding of N protein to boxB induces antitermination at λtR1 by preventing access of Rho to the rut sequences. This dual role was clearly shown in vivo by studying the effects of multiple mutations within the boxB hairpin stem on transcription termination and by substituting the N/boxB couple with the unrelated coat protein of phage MS2 and its stem-loop RNA binding site.