Introduction

Protein synthesis, or translation, is an inherently dynamic process in which the ribosome traverses the open reading frame of a messenger RNA (mRNA) template in steps of precisely one triplet-nucleotide codon, catalyzing the selection of aminoacyl-transfer RNA (aa-tRNA) substrates and polymerization of the nascent polypeptide chain, while simultaneously coordinating the sequential binding of exogenous translation factors. The complexity of this process is mirrored by the intricate molecular architecture of the ribosome itself, highlighted in atomic detail by recent X-ray crystallographic structures that reveal an elaborate network of RNA-RNA, RNA-protein, and protein-protein interactions (Korostelev and Noller, 2007; Steitz, 2008). This high degree of intra- and inter-molecular connectivity suggests that allosteric mechanisms may regulate the activity and coordinate the timing of biochemical events catalyzed by spatially distal ribosomal functional centers. Large-scale conformational dynamics of the ribosome have similarly been implicated as a means by which to regulate the biochemical steps of protein synthesis and to power forward progression through the kinetic steps of the translation process.

Comparison of X-ray crystallographic structures of ribosomal subunits as well as the intact ribosome in the absence and presence of translation factors (reviewed in Schmeing and Ramakrishnan [2009]), together with the analysis of cryogenic electron microscopy (cryo-EM) reconstructions of the ribosome trapped at various functional states during protein synthesis (see Chapter 7), has allowed visualization of large-scale conformational rearrangements of the translational machinery. Through such comparative structural analysis, mobile ribosomal domains have been identified and specific conformational changes have been inferred. However, these static structural images lack information regarding the timescales of the inferred conformational changes, and the kinetic and thermodynamic parameters underlying the corresponding ribosomal motions. Such dynamic information has recently been uncovered through the application of single-molecule fluorescence resonance energy transfer (smFRET) to studies of protein synthesis. This technique has proven to be particularly well-suited for monitoring and characterizing large-scale conformational dynamics of the ribosome and its tRNA and translation factor ligands, which often occur on length scales (∼tens of Ǻ) and time scales (∼ms to s) that are well matched with the spatio-temporal resolution of current smFRET methodologies (see Chapter 1). Guided by the structural data, numerous donor-acceptor fluorophore labeling schemes have already been developed, each capable of monitoring specific conformational changes of the translational machinery in real time.