Sequence elements essential for rho-dependent transcription termination at lambda tR1.

Abstract

To determine the location of DNA sequences required for the utilization of rho factor in transcription termination at the tR1 terminator of phage lambda, we constructed and analyzed a series of deletion mutants affecting several distinct regions of the cro-gene sequence. Two distinct sequence blocks, rutA and rutB, are shown to be particularly important for rho action at tR1 during in vitro transcription. They are located in the region between the sequence encoding the translation termination codon of cro mRNA and the start of tR1. Although other sequences contribute to rho action, their function can be replaced by unrelated heterologous DNA sequences encoding highly structured RNA segments, while the function of the rut sequences cannot. The lambda tR1 rut sequences encode RNA segments that contain a high proportion of cytidylate residues and that lack extensive intramolecular base pairing. Thus, the rut transcript segments are likely to be parts of the site on the nascent cro RNA that binds specifically with rho factor as an essential step in the termination process. This view is supported further by the findings (Chen, C.-Y. A., Galluppi, G. R., and Richardson, J. P. (1986) Cell 46, 1023-1028) that rho action at tR1 is specifically inhibited by DNA oligonucleotides that form hybrid helices with rut transcript segments. We also examined the effect of the auxiliary transcription factor NusA on transcription of our mutant templates and show that deletion of the boxA sequence of cro gene does not diminish the ability of NusA to reduce rho action at tR1.