A paused-backtracked intermediate in bacterial transcription initiation - lessons from single-molecule fluorescence spectroscopy

One of the main junctions of life and a central process in gene expression is DNA transcription. The transcription of RNA molecules from DNA by the RNA polymerase (RNAP) complex involves three main steps: initiaion, elongation & termination. Out of these, initiation is mostly the rate limiting step. When transcription initiates, RNAP finds the promoter, binds to it, melts 10-12 bases of duplex DNA, and then goes through multiple failed attempts to polymerize an RNA transcript. Using current models of how RNAP initiates transcription in bacteria, it was not possible to explain observed slow initiation kinetics. By performing a set of bulk and single-molecule experiments we have shown that transcription pausing, usually attributed only to transcription elongation, occurs also in initiation. We have shown that a paused-backtracked initiation intermediate is stabilized via a unique conformation. Here, we present an updated model of how transcription is initiated in bacteria, that includes the delay caused by pausing in initiation. The model that includes the pausing due to backtracking in transcription initiation, together with the associated conformational change, permitted providing at least one explanation of how RNAP might avoid inhibition by the antibiotic molecule, Rifampicin. Additional experiments have shown that indeed the discovered pausd-backtracked initiation intermediate allows avoiding transcriptional inhibition by Rifampicin per a given transcription cycle. Therefore, this finding serves as one (out of many possible other) mechanistic explanations for how bacteria might survive the inhibiting effects of antibiotics long enough until acquiring genetic-based resistance.