Analysis of the Intrinsic Transcription Termination Mechanism and Its Control

Abstract

Publisher Summary This chapter describes approaches that permit dissecting the termination process in several steps and identifying individual roles of the terminator elements, the hairpin and T-stretch, in each step. To study the basic mechanism of intrinsic termination and its control, an in vitro reconstituted system is used that contains Escherichia coli (E.coli) RNAP, a linear DNA template with a strong promoter and the intrinsic terminator, and pure elongation factors, NusA and N. E. coli NusA protein works as a general termination factor, which significantly increases the efficiency of many intrinsic terminators in vitro and in vivo. The trapped complex (TC) represents a unique configuration of the elongation complexes (EC) that occurs exclusively at the intrinsic termination points. Pausing at the tR2 termination point is determined by the downstream portion of its T-stretch. Pausing provides an additional time for the hairpin to form and is required for efficient termination under physiological conditions. To exclude the kinetic component (pausing) from the analysis of termination and to study the “mechanistic” component of the termination process in real time, the slow termination approach has been developed. This approach utilizes a modified A1-tR2 template carrying point substitutions within the T-stretch, rendering the RNA: DNA hybrid strong enough to delay the hairpin folding.