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.
Keywords: cell-free; ribosomes; transcription; translation; uncoupling.