A model for transcription termination suggested by studies on the trp attenuator in vitro using base analogs

Abstract

In vitro, termination of transcription at the attenuator site of the E. coli tryptophan ( rp) operon occurs efficiently in the absence of additional factors. In contrast, an altered attenuator site created by the deletion trpΔ LC1419 (which removes four of the eight consecutive TA base pairs at the termination site, but still functions partially in vivo) does not cause termination in vitro under standard conditions, even with the addition of rho factor. By varying components and conditions of the transcription reactions, we have found conditions under which termination does occur at the altered site ( trp a1419), as well as conditions affecting termination on the wild-type template. —RNA polymerase can terminate at trp a1419 when the analog iodo-CTP (which forms stronger base pairs with G) replaces CTP. —A mutant polymerase ( rpo203) that restores termination in strains carrying rho mutations can also terminate at trp a1419, at a position just past the deletion junction, suggesting that the point of termination may be specified by the remaining upstream leader sequence. —Analogs such as Br-UTP and allylamine-UTP, which form stronger base pairs with A, elevate readthrough levels at the wild-type attenuator site. —At low temperatures, readthrough on the wild-type template by wild-type RNA polymerase is enhanced, whereas the rpo203 enzyme still terminates efficiently. The results of these experiments suggest a model for termination of transcription in the absence of rho factor involving both RNA-RNA and RNA-DNA interactions. We propose that the response of RNA polymerase at a terminator site is governed by a balance between the formation or stability of a hairpin structure in the transcript and the strength of polymerase-stabilized base pairing between the distal nucleotides of the transcript and the DNA template. In more complex cases in vitro, or within the cell, formation or stabilization of these structures may also involve other regions of RNA and additional factors.