PT - JOURNAL ARTICLE AU - Lea L. de Maddalena AU - Henrike Niederholtmeyer AU - Matti Turtola AU - Zoe N. Swank AU - Georgiy A. Belogurov AU - Sebastian J. Maerkl TI - GreA and GreB enhance <em>Escherichia coli</em> RNA polymerase transcription rate in a reconstituted transcription-translation system AID - 10.1101/024604 DP - 2016 Jan 01 TA - bioRxiv PG - 024604 4099 - http://biorxiv.org/content/early/2016/01/18/024604.short 4100 - http://biorxiv.org/content/early/2016/01/18/024604.full AB - 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. 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. Graphical abstract