Abstract
Decades of traditional biochemistry, structural approaches, and, more recently, single-molecule-based in vitro techniques have provided us with an astonishingly detailed understanding of the molecular mechanism of ribosome-catalyzed protein synthesis. However, in order to understand these details in the context of cell physiology and population biology, new techniques to probe the dynamics of molecular processes inside the cell are needed. Recent years' development in super-resolved fluorescence microscopy has revolutionized imaging of intracellular processes, and we now have the possibility to directly peek into the microcosm of biomolecules in their native environment. In this Perspective, we discuss how these methods are currently being applied and further developed to study the kinetics of protein synthesis directly inside living cells.
Highlights
Decades of traditional biochemistry, structural approaches, and, more recently, single-molecule-based in vitro techniques have provided us with an astonishingly detailed understanding of the molecular mechanism of ribosomecatalyzed protein synthesis
The core functions of ribosomes are highly conserved throughout the tree of life, where the major differences between prokaryotic and eukaryotic mRNA translation lie in the highly regulated steps of translation initiation, i.e., when the small and large ribosomal subunits assemble on the start codon of the mRNA to initiate polypeptide synthesis
We have a very limited understanding of the dynamic interplay between the protein synthesis machinery and other intracellular pathways, and we still lack quantitative models to connect the molecular details to cell physiology and population biology
Summary
Structural approaches, and, more recently, single-molecule-based in vitro techniques have provided us with an astonishingly detailed understanding of the molecular mechanism of ribosomecatalyzed protein synthesis. The RNA-binding proteins and peptide-binding antibodies were either labeled by organic dyes in vitro and microinjected into the cells or expressed in the cells as genetic fusions to FPs. Compared to a simple expression of fluorescent proteins, which due to the maturation time produces a delay between translation and detection,[21] the use of preformed tag-specific fluorophores allowed immediate readout of translation activity.
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