Molecular Movement inside the Translational Engine

of the translational elongation cycle. The original twoTranslation requires iterative coupled movement of mRNA site mechanism for elongation, proposed by Watson and tRNA throughout the elongation phase of protein over 30 years ago, was elaborated by the discovery of synthesis. Each new amino acid is recruited to the riboa third site, called the exit, or E site (Rheinberger et some as an aminoacyl-tRNA·EF-Tu·GTP ternary comal., 1981), and many of the details of the model were plex. Following peptide bond formation, the tRNAs and confirmed or extended. The three-site version of the associated mRNA must be translocated from one riboclassical model is summarized schematically in Figure somal site to the next in a GTP-dependent process that 1. Beginning with an initiator or peptidyl tRNA in the P is catalyzed by elongation factor EF-G. On a molecular (peptidyl) site (Figure 1A), a new aminoacyl tRNA, with scale, this movement is substantial, involving excuran anticodon that is complementary to the available A sions on the order of 50 Å at the elbow of tRNA during (aminoacyl)-site codon, is introduced as an aminoacyleach elongation step. Although the elongation cycle retRNA·EF-Tu·GTP ternary complex. Following hydrolysis quires the two G proteins (elongation factors EF-Tu and of GTP and release of EF-Tu, the aminoacyl-tRNA is EF-G) under physiological conditions, it has been shown bound to the A site (Figure 1B). The anticodon ends that protein synthesis can becarried out by the ribosome of both tRNAs interact with the 30S subunit, and their itself, in the absence of factors, or GTP, under certain acceptor (aminoacyl) ends interact with the 50S subunit. in vitro conditions (Pestka, 1969; Gavrilova et al., 1976). Attack of the peptidyl-tRNA bond by the a-amino group Thus, the ability to move mRNA and tRNA is an inherent of aminoacyl-tRNA, a spontaneous reaction catalyzed property of the ribosome; the factors serve to increase by peptidyl transferase (an activity of the 50S subunit), the speed and accuracy of elongation in a GTP-depenresults in peptide bond formation and transfer of the dent manner. The ribosome can therefore be considered growing peptide chain to the A-site tRNA (Figure 1C). as a macromolecular machine. Movement of the newly created peptidyl-tRNA from the Because of the fundamental importance of translation A to P site is accomplished by EF-G in a GTP-dependent to all life as we know it, and the essential similarities reaction (Figure 1D). At the same time, the deacylated between all ribosomes, the movement associated with tRNA moves to theE site. The E site ismost likely located the translational elongation cycle must be one of the exclusively on the 50S subunit (Kirillov et al., 1983; Lill most ancient and basic in biology. Understanding the and Wintermeyer, 1987; Moazed and Noller, 1989a); this underlying molecular basis of this movement has prewould mean that theelaborated elongation cycle is really sented a formidablechallenge togenerations of molecua two-and-a-half-site, rather than a three-site, model. lar biologists. The ribosome is large (about 2.5 MDa) Deacylated tRNA, bound weakly to the E site, dissociates and structurally complex (more than 50 different profrom the ribosome to complete the cycle of elongation. teins and three RNA molecules comprising over 4500 It has been proposed that binding of the next aminoacylnucleotides; Hill et al., 1990; Matheson et al., 1995). It tRNA to the A site allosterically weakens the affinity of is also functionally complex. It is divided into a small tRNA for the E site (Nierhaus, 1990), but this has recently and a large subunit, which in bacteria are called the been challenged (Semenkov et al., 1996). 30S and 50S subunits. In addition to movement, the Chemical footprinting studies showed that tRNAs ribosome must provide specific binding sites for mRNA, bound in their various ribosomal binding states protect tRNA, and the various initiation, elongation, and terminacharacteristic bases in rRNA from chemical probes, protion factors, and catalyze peptide bond formation. It viding structural correlates for the states of tRNA during must also stabilize codon–anticodon interaction and the elongation cycle (Moazed and Noller, 1986, 1989a). preserve the translational reading frame. It was found that, in certain intermediate states of elonIn recent years, many fundamental assumptions congation, the two ends of a tRNA could be in different cerning the translational elongation cycle have been states on the two ribosomal subunits; for example, a challenged, and some have required drastic revision. In tRNA could simultaneously occupy the 30S A site and this article, we focus on the process of translocation— the 50S P site. Interpretation of these experiments rethe precisely orchestrated movement of tRNA from one sulted in the hybrid states model for the elongation cyribosomal site to the next, coupled to movement of