Single-Molecule Tracking Approaches to Protein Synthesis Kinetics in Living Cells

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.

A Ribosomal Perspective on Neuronal Local Protein Synthesis

“…But, these approaches provide very little dynamic information in the cellular realm. More recent studies based on single-molecule tracking have been instrumental in describing the dynamic properties of ribosomes and mRNAs within cells ( Volkov and Johansson, 2019 ). Both prokaryotic and eukaryotic ribosomes have been visualized by tagging RPs with fluorescent proteins ( Katz et al, 2016 ; Bayas et al, 2018 ).…”

“…These experiments have been critical in understanding the population behavior of ribosomes arising from thousands of single-molecule detection events. Especially, live tracking of ribosomal subunits allows correlating their dynamics to mRNA translation within a cell ( Prabhakar et al, 2019 ; Volkov and Johansson, 2019 ). For example, single-molecule diffusion studies on E. coli ribosomes have found that while the subunits themselves can diffuse freely across the entire cell, elongating ribosomes are excluded from the nucleoid ( Sanamrad et al, 2014 ).…”

“…These results suggest that polyribosomes impede the movement of mRNP complexes by making them heavier while destabilizing them with puromycin increases their mobility ( Katz et al, 2016 ). However, one problem with single-molecule experiments is that the fluorescent proteins coexpressed with the proteins of interest often show moderate brightness and photostability ( Chudakov et al, 2010 ; Volkov and Johansson, 2019 ). In addition, the presence of a large number of subdiffraction ribosomal particles can complicate the detection and analysis of single-molecule trajectories ( Volkov and Johansson, 2019 ).…”

Quantitative Modeling of Protein Synthesis Using Ribosome Profiling Data

“…Single-molecule experiments provided significant insights into the details of the process of protein synthesis ( Munro et al, 2008 ; Volkov and Johansson, 2018 ). However, little was known on how different mRNA features can control protein synthesis until the Next Generation Sequencing (NGS) experiments started uncovering various kinetic properties of this process ( Ingolia et al, 2019 ).…”

Rational Design of Aptamer-Tagged tRNAs

“…Although several types of tRNA variants fused with hairpin RNA aptamers have been developed [ 7 , 8 , 9 , 10 , 11 , 12 , 13 ], these fusion RNAs usually lost the translational activity of tRNA. Recently, “V-Spinach tRNAs” were developed by inserting a Baby Spinach aptamer [ 14 ] into the V-loop of a few E. coli tRNA species [ 8 ], although the V-Spinach tRNAs showed only marginal tRNA activities [ 8 , 15 ]. More recently, the V-arm hairpin of a yeast serine tRNA species was replaced by the MS2 phage hairpin RNA (19 nucleotides in length) to recruit an enzyme fused with the MS2 coat protein on the premature form of these fusion RNA molecules in yeast cells [ 11 ].…”