Pausing in Escherichia Coli transcription initiation
An essential and highly regulated step in gene expression is transcription initiation. After promoter binding and DNA unwinding (‘bubble opening’) and in the presence of nucleoside triphosphates (NTPs), the RNA polymerase (RNAP)-promoter initial transcribing complex (RPitc) engages in ‘abortive initiation’, a process in which RNAP cycles between synthesis and release of short RNA transcripts. In abortive initiation, RPitc is believed to undergo a sequence of transitions between different initiation sub-states. The kinetics of the production of a full RNA transcript starting at a late initiation sub-state is expected to be similar or faster than the kinetics measured from an earlier initiation sub-state. To test this hypothesis, we developed a novel in vitro single-run quenched kinetics transcription assay based on the detection and quantification of run-off transcripts. Using this assay and corroborating it with gel-based and magnetic tweezer assays, testing two different promoters, we surprisingly found that run-off transcription kinetics starting from late initiation sub-states is slower than kinetics starting from earlier initiation sub-states. When the same kinetic measurements were performed in the presence of the transcription elongation factor GreA, the kinetics starting from a late initiation sub-state was accelerated.
Experimental results suggest that as a function of shortage in NTPs RPitc can enter an off-pathway state in which the nascent RNA is in a backtracked, paused position, awaiting incorporation of the missing NTP. Our findings suggest that pausing at distinct stages of transcription initiation could regulate gene expression under stressed conditions.