rut Sites in the nascent transcript mediate Rho-dependent transcription termination in vivo

The in vitro function of the coliphage lambda tR1 Rho-dependent terminator is governed primarily by a tripartite upstream sequence element designated rut. To determine the contribution of the different components of the rut site to terminator function in the normal context of coupled translation of the nascent cro message, tR1 variants lacking different rut site sequences were tested for terminator function in vivo. Intact rutA and rutB sequences were both necessary for efficient termination. However, deletion of the upstream rutA was far more detrimental than deletion of rutB. The intervening boxB, which encodes a short RNA stem and loop, could be deleted without reducing termination or detectably altering Rho's interaction with the corresponding cro transcript. The relative importance of these sequence elements was also the same in a minimal in vitro termination assay system. Rut sequences are therefore essential for terminator function in vivo and rutA contributes substantially more to tR1 function than does rutB. The relative contribution of these elements can be ascribed to differences in Rho's binding affinity for the encoded transcripts. If other cellular factors also bind the rut element RNA, they do not alter the relative contribution of its two regions to Rho-dependent transcription termination in vivo.