MULTIPLE INTERACTIONS STABILIZE A SINGLE PAUSED TRANSCRIPTION INTERMEDIATE IN WHICH HAIRPIN TO 3' END SPACING DISTINGUISHES PAUSE AND TERMINATION PATHWAYS

Transcription is delayed in the leader regions of the Escherichia coli trp and his operons by multipartite pause signals that consist of fou r components: a nascent RNA structure (the pause hairpin), the 10 or 1 1 nt 3'-proximal region between the pause hairpin and the RNA 3' end, the bases in the active site, and similar to 14 bp of duplex DNA downs tream from the pause site. Results described in the accompanying paper suggest that the Iris pause hairpin slows nucleotide addition via int eraction with an easily disordered surface on RNA polymerase. Here we report that the four pause signal components slow nucleotide addition in a single kinetic intermediate. Formation of tile paused transcripti on complex, in contrast, involves synergistic effects of RNA and DNA s equences that select the wild-type pause site from among several adjac ent possibilities. Extending the pause hairpin with one G.C base-pair reduces pausing, apparently by interfering with pause hair-pin interac tion; adding a second C.G base-pair that reduces the 3'-proximal RNA t o 9 nt or less (within the 7 to 9 nt characteristic of rho-independent terminators) induces transcript release. We propose that escape from the pause is governed by a rate-limiting isomerization that may requir e substrate NTP binding to re-establish the active site geometry, wher eas transcript release and termination ensue when the hairpin interact ion is weakened and isomerization to an active conformation is blocked . (C) 1997 Academic Press Limited.