ESCHERICHIA-COLI RNA-POLYMERASE TERMINATES TRANSCRIPTION EFFICIENTLY AT RHO-INDEPENDENT TERMINATORS ON SINGLE-STRANDED-DNA TEMPLATES

Several models have bi en proposed for the mechanism of transcript: te rmination Escherichia coli RNA polymerase at rho-independent terminato rs, Yager and von Hippel (Yager, T. D. & von Hippel, P. H. (1991) Bioc hemistry 30, 1097-118) postulated that the transcription complex is st abilized by enzyme-nucleic acid interactions and the favorable free en ergy of a 12-bp RNA-DNA hybrid but is destabilized by the free energy required to maintain an extended transcription bubble. Termination, by their model, is viewed simply as displacement of the RNA transcript f rom the hybrid helix by reformation of the DNA helix. We have proposed an alternative model where the RNA transcript is stably bound to RNA polymerase primarily through interact ions with two single-strand spec ific RNA-binding sites: termination is triggered by formation of an RN A hairpin that reduces binding of the RNA to one RNA-binding site and, ultimately leads to its ejection from the complex, To distinguish bet ween these models, we have tested whether E. coli RNA polymerase can t erminate transcription at rho-independent terminators on single-strand ed DNA. RNA polymerase cannot form a transcription bubble on these tem plates; thus, the Yager-von Hippel model predicts that intrinsic termi nation will nor occur. We find that transcript elongation on single-st randed DNA templates is hindered somewhat by DNA secondary structure. However, E. coli RNA polymerase efficiently terminates and releases tr anscripts at several rho-independent terminators on such templates at: the same positions as termination occurs on duplex DNAs, Therefore, n either the nontranscribed DNA strand nor the transcription bubble is e ssential for rho-independent termination by E. coli RNA polymerase.