Rho-independent termination: dyad symmetry in DNA causes RNA polymerase to pause during transcription in vitro

Abstract

Termination of transcription by RNA polymerase at rho-independent sites appears to depend primarily upon two structural features, a region of GC-rich dyad symmetry in the DNA preceding the stop point and a stretch of uridines at the 3' end of the transcript. The possibility that the former might be responsible for slowing elongation prompted us to perform a kinetic analysis of transcription across the leader and terminator regions of the E. coli tryptophan (trp) operon. Regions where the elongation rate is dramatically slowed or stopped are identifiable because they generate discrete transcript hands on a gel. Species derived from pause sites, unlike those resulting from termination sites, are transient and detectable only within the first two minutes of transcription, since polymerase eventually resumes elongation. At two mutant trp attenuator sites (trp a135 and trp a1419), where termination is incomplete or absent in vitro, a substantial pause is nevertheless observed. Likewise, a significant pause occurs at trp t, the termination site at the end of the operon. Our experiments also reveal a major pause site at about position 90 in the trp leader sequence, just past a region of dyad symmetry. The RNA hairpin corresponding to this site is U-rich, and pausing is strongly enhanced by incorporation of BrUTP. In contrast, this analog does not affect pausing at the attenuator or terminator sites with hairpins that are GC-rich. These results strongly support the hypothesis that pausing of the polymerase is an obligatory prelude to rho-independent termination. Moreover, the termination event evidently results from consecutive but discrete responses to separate structural features of these sites.