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

Abstract

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.

Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness

“…Recently, studies in Salmonella enterica serovar Typhimurium demonstrated that GreA-mediated rescue of backtracked paused complexes during transcription is crucial in promoting hilD expression through its 3 -UTR (Gaviria-Cantin et al, 2017). In addition, GreA enhanced expression of several E. coli RNAP promoters in vitro (Erie et al, 1993;Feng et al, 1994;Hsu et al, 1995;Maddalena et al, 2016). Given that GreA has a multitude of effects on transcription, initiation, and elongation (Stepanova et al, 2007;Vinella et al, 2012), this could explain our failure to identify the binding motif of GreA.…”

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

“…(C) Optimization of the system can be carried out by adjusting both protein and energy solution components. Potential system modifications are shown: (D) supplementation with E. coli RNAP allows for more complex transcription regulation (Maddalena et al, 2016); (E) addition of chaperones aids protein folding (Niwa et al, 2012); (F) vesicles enable membrane protein folding and assembly (Kuruma and Ueda, 2015;Niwa et al, 2015a;Jacobs et al, 2019); and (G) oxidizing conditions allow for disulfide bond formation (Shimizu et al, 2005). (Tuckey et al, 2014), and Magic PURE system (Creative Biolabs).…”

“…This improvement lowers the price of the energy source and simplifies the energy regeneration process (Wang et al, 2019). While the original PURE system only contains T7 RNA polymerase, with its limited capability for transcriptional regulation, E. coli σ -factor based transcription has been successfully demonstrated, albeit with low efficiency with certain promoters, which can be enhanced by adding purified E. coli polymerase alone or in combination with transcription elongation factors (Maddalena et al, 2016) (Figure 2D).…”

Cell-free translation is more variable than transcription

“…16 A likely explanation for this discrepancy is the decoupling of RNA and protein synthesis when using T7 RNA polymerase and E. coli ribosomes. 1,16,25 Perhaps substituting T7 RNA polymerase with E. coli RNA polymerase, as in ePURE, 34 would allow for proper coupling and thus the ability to predictably control protein output through the activity of the transcriptional promoter.…”