Viewing folding of nascent polypeptide chains from ribosomes

Abstract

Regulation of gene expression occurs at multiple stages and ultimately determines the protein levels in cells. The last decade has witnessed impressive progress in the development of approaches to measuring changes in cellular transcription and protein complement. However, techniques monitoring protein synthesis have lagged far behind. Current proteomic studies gauge the steady state levels of intracellular protein and rely on mass spectrometry, which has a low dynamic range and rarely samples more than 10% of the proteome [1]. RNA-seq is a sensitive approach for assessing transcriptional changes but the transcriptome correlates poorly with changes in protein levels [2,3]. Recent data suggest that the regulation of gene expression within cells occurs predominantly at the level of translation and that protein abundance correlates closely with translation rates [3]. Therefore, monitoring protein synthesis is crucial to understanding cellular homeostasis. Additionally, regulation of protein synthesis at the level of translation permits the cell to respond swiftly to adverse conditions [4]. Therefore, translational control is an integral part of cellular stress response. An intricate process associated with protein synthesis is folding. Proteins must achieve proper tertiary structure to acquire their designated properties and functions. Our current understanding of protein folding is predominantly based on in vitro refolding of denatured full-length proteins [5]. However, under native intracellular conditions, protein folding could occur concurrently with the synthesis of primary polypeptide chains on the ribosomes [6,7]. Cotranslational folding of partially synthesized nascent chains differs from refolding of full length polypeptides owing to the vectorial nature and the relatively slow elongation speed of the translation process. However, it is challenging to capture cotranslational folding events of nascent chains attached to the translating ribosome. Several approaches have been developed to dissect this process but most of them rely on in vitro systems with limited resolution [7,8]. There is thus an urgent need to establish new approaches to monitor cotranslational folding processes in vivo with high resolution. One solution is to look at the folding status of nascent polypeptide chains from the perspective of translating ribosomes.