Investigating the consequences of mRNA modifications on protein synthesis using in vitro translation assays.

The ribosome translates the information stored in the genetic code into functional proteins. In this process messenger RNAs (mRNAs) serve as templates for the ribosome, ensuring that amino acids are linked together in the correct order. Chemical modifications to mRNA nucleosides have the potential to influence the rate and accuracy of protein synthesis. Here, we present an in vitro Escherichia coli translation system utilizing highly purified components to directly investigate the impact of mRNA modifications on the speed and accuracy of the ribosome. This system can be used to gain insights into how individual chemical modifications influence translation on the molecular level. While the fully reconstituted system described in this chapter requires a lengthy time investment to prepare experimental materials, it is highly verstaile and enables the systematic assessment of how single variables influence protein synthesis by the ribosome.