Luciferase-based reporter system for in vitro evaluation of elongation rate and processivity of ribosomes

The elongation step of translation is a key contributor to the abundance, folding and quality of proteins and to the stability of mRNA. However, control over translation elongation has not been thoroughly investigated. In this study, a Renilla-firefly luciferase fusion reporter system was further developed to investigate the in vitro elongation rate and processivity of ribosomes independent of the initiation and termination steps. The reporter mRNA was constructed to contain a single ORF encoding in-frame Renilla luciferase, a specific domain moiety and firefly luciferase. Such a reporter structure enables the quantitative and individual evaluation of the synthesis of a specific domain. As a proof of principle, the synthesis of three protein domains of different lengths and structures was analyzed. Using a cell-free translation assay, both the elongation rate and processivity of ribosomes were shown to vary depending on the domain synthesized. Additionally, a stalling sequence consisting of ten rare arginine codons notably reduced the elongation rate and the processivity of the ribosomes. All these results are consistent with the previously known dynamics of elongation in vivo. Overall, the methodology presented in this report provides a framework for studying aspects that contribute to the elongation step of translation.

Automated summary

The study developed a Renilla-firefly luciferase fusion reporter system to investigate ribosome elongation rate and processivity independent of initiation and termination steps. Using a cell-free translation assay, it was shown that elongation rate and processivity of ribosomes vary depending on the domain being synthesized, and a stalling sequence of rare arginine codons notably reduced ribosome elongation rate and processivity. These results are consistent with known dynamics of elongation in vivo, providing a framework for studying translation elongation.