Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro

ABSTRACT The conventional view regarding regulation of gene expression is based on transcription control. However, a growing number of recent studies has revealed the important additional impact of translational regulation. Eukaryotic translational machinery appears to be capable of reprogramming mRNA translation to generate proteins required to maintain a healthy cellular proteostasis under particular physiological conditions or to adapt to stress. Although the mechanisms of such remarkable regulation are beginning to emerge, recent studies have identified the ribosome as one of the major constituents of translation-dependent control of gene expression that is especially important during stress. Built of RNA and proteins, ribosomes are susceptible to environmental and intracellular stresses. How stress-modified ribosomes regulate translation and whether they play a role in stress-induced gene expression remain largely elusive. This knowledge gap is likely due to the lack of an appropriate experimental system. Canonical approaches based on exposing cells or cell-free extracts to stressors provide inconclusive results due to off-target effects of modifying agents. Here we describe a robust and simple in vitro assay that allows separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution of the translation reaction. This r ibosome s eparation and r econstitution assay (RSR) is highly advantageous, as it allows modification of ribosomes without compromising other key translational components, followed by supplementing the ribosomes back into translation reactions containing undamaged, translationally-competent yeast lysate. Besides addressing the impact of ribosome-derived stress on translation, RSR can also be used to characterize mutated ribosomes and ribosomes devoid of associated factors.