Insights into Elongation Dynamics in Human Translation from a Fully Reconstituted In Vitro System
Vaishali Goyal · 2026
Non-uniform local rates of mRNA decoding by ribosomes are a hallmark of translation elongation. Transient ribosome pausing during nascent peptide elongation assists in the proper folding and maturation of proteins. The local rate of translation is modulated by the position of ribosome on the mRNA, the composition of the nascent peptide, and its interaction with the ribosome and ribosome-associated protein biogenesis. We aimed to study the kinetics of translation during early stages of elongation in humans with a focus on the role of auxiliary translation factors and molecular chaperones in modulating this process. To that end, we established a fully reconstituted in vitro human translation system comprising purified 40S and 60S ribosomal subunits, essential initiation and elongation factors, and human aminoacyl-tRNAs. We optimized our in vitro system to achieve peptide synthesis rates comparable to those observed in vivo, thereby enabling real-time monitoring of peptide elongation dynamics and the identification of transient translational pauses during elongation. Notably, some of these pauses were associated with polyproline stretches and were alleviated upon the addition of eIF5A, resulting in 2.5-fold increase in the translation rate. We also observed that the formation of the first peptide bond occurs significantly more slowly than the average rate of nascent peptide elongation. Strikingly, pausing at early translation intermediates was prolonged in the presence of the co-translational chaperone nascent polypeptide-associated complex (NAC). Investigation of the underlying mechanism revealed that NAC-induced pausing does not require direct interaction between NAC and the nascent peptide but instead occurs indirectly through the interaction of NAC with the ribosome. These findings open intriguing possibilities for allosteric regulation of local translation rates by ribosome-associated protein biogenesis factors such as NAC. In summary, we have established a robust and versatile in vitro system for studying ribosome pausing during translation elongation in humans, which can be fine-tuned to uncover the molecular mechanisms underlying this poorly understood aspect of translational control.