Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems

Secondary structure formation of mRNA, caused by desynchronization of transcription and translation, is known to impact gene expression in vivo. Yet, inactivation of mRNA by secondary structures in cell-free protein expression is frequently overlooked. Transcription and translation rates are often not highly synchronized in cell-free expression systems, leading to a temporal mismatch between the processes and a drop in efficiency of protein production. By devising a cell-free gene expression platform in which transcriptional and translational elongation are successfully performed independently, we determine that sequence-dependent mRNA secondary structures are the main cause of mRNA inactivation in in vitro gene expression.