Single-Molecule Fluorescence Applied to Translation.

Single-Molecule Fluorescence Applied to Translation Arjun Prabhakar,1,2 Elisabetta Viani Puglisi,1 and Joseph D. Puglisi1 1 Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 2 Program in Biophysics, Stanford University, Stanford, California 94305 Correspondence: Single-molecule fluorescence methods have illuminated the dynamics of the translational machinery. Structural and bulk biochemical experiments have provided detailed atomic and global mechanistic views of translation, respectively. Single-molecule studies of translation have bridged these views by temporally connecting the conformational and compositional states defined from structural data within the mechanistic framework of translation produced from biochemical studies. Here, we discuss the context for applying different single-molecule fluorescence experiments, and present recent applications to studying prokaryotic and eukaryotic translation. We underscore the power of observing single translating ribosomes to delineate and sort complex mechanistic pathways during initiation and elongation, and discuss future applications of current and improved technologies. P roteins must be translated rapidly, with high fidelity and quality by the ribosome. Translation is thus a highly dynamic process with temporal changes in ligand stoichiometry on the ribosome, and conformational changes within ribosomes and ligands (Noller et al. 2017a,b; Rodnina et al. 2017). During initiation in all organisms (Merrick and Pavitt 2018; Rodnina 2018), ribosomal subunits must assemble at the correct start site on the messenger RNA (mRNA) defined by specific mRNA–ribosome interactions; this establishes the reading frame for translation of the correct protein sequence. On proper initiation, the assembled 70S or 80S ribosome with initiator transfer RNA (tRNA) base-paired with the start codon positioned in the peptidyl-tRNA site (P site) is competent for elongation of the polypeptide. During elonga- tion (Dever et al. 2018; Rodnina 2018), the genetic code is read by cognate aminoacyl-tRNAs delivered to the aminoacyl-tRNA site (A site) as a complex with a GTPase elongation factor (EF-Tu in bacteria). This dynamic process of tRNA selection occurs with high fidelity (one wrong amino acid incorporated per 3000) (Loftfield 1963), aided by the free energy of GTP hydrolysis, ribosome and factor conformational changes, and induced fit with kinetic proofreading. Once a correct tRNA is accommodated into the peptidyl transferase center (PTC) of the ribosome, peptide bond formation occurs with subsequent conformational changes in the ribosome (intersubunit rotation) and tRNAs (hybrid state formation). The codon–anticodon pairs on the ribosome must be precisely and rapidly moved from the P and A sites to the Editors: Michael B. Mathews, Nahum Sonenberg, and John W.B. Hershey Additional Perspectives on Translation Mechanisms and Control available at www.cshperspectives.org Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a032714 Cite this article as Cold Spring Harb Perspect Biol 2019;11:a032714 1 A. Prabhakar et al. tion complex demands coordinated binding and catalysis of many protein factors triggered by the stop codon signal (Petry et al. 2008; Sternberg et al. 2009; Dever and Green 2012; Koutmou et al. 2014; Peske et al. 2014; Prabhakar et al. 2017a). Underlying the process of translation are conformational dynamics of the ribosome and macromolecules that guide translation (Prabhakar et al. 2017b). Layered onto the basal process are regulatory events that modulate temporally the output of translation—the nature, rates, and efficiency of production of the protein product. An understanding of dynamics is thus required to delineate the mechanism of translation. Traditional study of dynamics involves measurements in bulk on large numbers of molecules in solution (Wintermeyer et al. 2004; Johansson et al. 2008). Chemical and biochemical kinetics methods have provided the lion’s share of information on the time evolution of translation. Reactions are initiated by rapid mixing of the components, and a time-dependent signal is measured in a stopped-flow system either (1) in real time (e.g., fluorescence) to report compositional and conformational changes (Rodnina et al. 1994; Studer et al. 2003; Koutmou et al. 2014), or (2) using quenched-flow methods recording a chemical signal as a function of time to report chemical changes ( peptide bond formation, GTP or ATP hydrolysis) (Bilgin et al. 1992; exit (E site) and P site during the process of translocation, catalyzed by another GTPase elongation factor (EF-G in bacteria), which also resets the conformation of the ribosomal subunit and places the next codon in the A site. This elongation cycle repeats until a stop codon enters the A site, which signals release factors (RFs) to catalyze release of the polypeptide from the P-site tRNA during termination, and then prompts the ribosomal subunits to be split during recycling (Hellen 2018). PROBING THE DYNAMICS OF TRANSLATION The basal process of translation is thus highly directional and dynamic. Even after the process of ribosome biogenesis, whereby ribosomal RNAs are transcribed, processed, and assembled with ribosomal proteins (Shajani et al. 2011), the ribosome undergoes numerous changes in conformation and composition during translation (Fig. 1). Ribosomal subunits assemble during initiation; tRNAs flux through the ribosome as it traverses a messenger RNA (mRNA) (Uemura et al. 2010), unfolding potential RNA structures (Wen et al. 2008), while spooling out a growing nascent polypeptide (which also may fold cotranslationally) during elongation (Thommen et al. 2017); and the disassembly of this transla- Elongation Cotranslational protein folding Protein factor binding/dissociation tRNA flux Nascent peptide interactions Termination/recycling Initiation mRNA structure 50S tRNA mRNA Assembly Ribosome domain movements Ribosome procession Disassembly 30S Figure 1. Dynamic picture of translation. Translation is regulated through a complex network of interactions and movements of the ribosomal subunits, the translated messenger RNA (mRNA), the adaptor transfer RNAs (tRNAs), protein translation factors, and the growing nascent peptide chain. Key dynamic features of translation are highlighted. 2 Cite this article as Cold Spring Harb Perspect Biol 2019;11:a032714 Single-Molecule Fluorescence Applied to Translation Pape et al. 1998). By using these approaches, the basic kinetic mechanisms of initiation (Antoun et al. 2003; Milon et al. 2012), elongation (Gromadski et al. 2002; Johansson et al. 2008), and termination/recycling (Karimi et al. 1999; Zavialov et al. 2001; Peske et al. 2005, 2014; Koutmou et al. 2014) in bacteria have been determined, and progress in understanding the special features of eukaryotic translation made (Shoemaker and Green (...truncated)