NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble

Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.</p

GreA and GreB Enhance Expression of Escherichia coil RNA Polymerase Promoters in a Reconstituted Transcription-Translation System

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate of Escherichia coli RNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of natural E. coli promoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and synthetic E. coli promoters and characterization of their regulation in minimal and defined reaction conditions, making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology