Rho-dependent transcription termination at lambda R1 requires upstream sequences.

In order to define the DNA sequence requirements for rho-dependent transcription termination, we have constructed and analyzed two classes of mutants affecting the cro-tR1 region of phage lambda: one class contains progressively larger deletions in the region upstream of the rho-dependent terminator tR1, and the other consists of small insertions or deletions generated by the linker scanning procedure. We show that rho-dependent termination at lambda tR1 in vitro requires contribution from sequences far upstream of the sites of termination. No single linker scanning mutant completely abolishes transcription termination, indicating that there exists no unique sequence that serves as an obligatory rho utilization site. Our results suggest that rho can interact with a large portion of the RNA transcript, including sequences that would be translated in vivo. Moreover, rho action appears to require a minimal transcript length, about 150-190 nucleotides for the tR1 termination sites. We also examine the effect of the auxiliary transcription factor NusA on transcription of these mutants, and we provide support for the proposal that specific effects of NusA on transcription termination require a sequence known as "boxA" (Olson, E.R., Flamm, E.L., and Friedman, D.I. (1982) Cell 31, 61-70).