Christian M. Kaiser and Ignacio Tinoco , Jr.
In this Review, we will describe the application of single-molecule approaches to study the translation of mRNA by single ribosomes, focusing on mechanical manipulation with optical tweezers. Single-molecule methods have distinct advantages over traditional ensemble (or bulk) experiments in identifying the molecular mechanisms underlying this complex biological process. This has particularly important implications for elucidating the kinetics of processive molecular machines, such as ribosomes. The kinetics contain abundant information about how molecular machines act in a coordinated fashion to accomplish a complicated task, such as deciphering a nucleic acid sequence and synthesizing the encoded protein. However, kinetics are governed by stochastic processes; thus, each molecule in a reaction takes a different amount of time to react. It is impossible to maintain synchronicity over several steps of a sequential reaction. At any time in an ensemble reaction of many molecules, there will be reactants, products, and all of the intermediates. For a large number of ribosomes that start translating a particular mRNA at the same time, some will be decoding the first codon, while others are reading the second, third, fourth, etc. In contrast, at any time in a single-molecule reaction, there is only one species. The characteristics of each single species can be determined.
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