Probing the mechanisms of translation with force.

Genetic information flows from DNA to RNA to protein. DNA is transcribed to yield messenger RNAs (the coding RNA), RNAs required for protein synthesis (ribosomal RNAs and transfer RNAs), and non-coding RNAs that often have regulatory functions (ncRNAs, including microRNAs, siRNAs, piRNAs,1 and others). Translation of messenger RNAs (mRNAs) produces the proteins that characterize the structures and functions of all organisms from bacteria to humans. At the core of the translation machinery is the ribosome, a macromolecular complex comprised of over 50 proteins and three ribosomal RNAs with a molecular weight of 2.5 MD for bacteria and 4.3 MD for humans.2 The ribosome cooperates with auxiliary species to ensure accurate and highly regulated translation. These species include initiation factors, elongation factors, transfer RNAs with attached amino acids, as well as release and recycling factors.3,4 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 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. Optical tweezers have been used to study translation by a single ribosome on one mRNA. In the first published study, constant force was applied to the 3’- and 5’-ends of a harping RNA.5 As the ribosome translated the hairpin, double strand RNA was converted to single strands, thus increasing the end-to-end distance of the RNA molecule. The increase in distance directly measures the translocation of the ribosome. In a subsequent set of experiments, force is applied to either the 3’- or 5-end of the RNA while holding on to the ribosome. This geometry allows studying the ribosome as a motor under assisting force or opposing force as it moves along its track (the mRNA). A third geometry has been developed in which force is applied across the nascent polypeptide. This experimental setup will allow synthesis of the translation product to be monitored. 1.1 The Machinery of Translation High resolution structures of the ribosomal subunits6-12 as well as the 70S ribosome13 have been obtained by X-ray diffraction around the year 2000. A thorough review (with over 80 references) of the structural aspects of the functions of the ribosome is given at the Nobel Prize 2009 website (http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2009/advanced.html). Further details are described by each Nobelist in their published Nobel lectures.14-16 The structures of bacterial ribosomes complexed with mRNAs, tRNAs, initiation, elongation, and release factors, and antibiotics are presented. More recent atomic resolution structures include a 3.0 A structure of a yeast eukaryotic ribosome,17-19 the human and Drosophila 80S ribosome 20, release factor 2 and 3 bound to a bacterial ribosome,21-23 and EF-G bound to the ribosome.24-27 Cryo-electron microscopy, cryo-EM, provides further information about different states of the ribosome and its interactions with external factors.28-31 Structural information of many of the auxiliary species, free and bound to the ribosome are now available: initiation factors, eIF1;32 elongation factors, EF-G,33,34 EF-Tu,35-39, as well as release and recycling factors.40-43