- Open Access
Toward an understanding of the structural basis of translation
© BioMed Central Ltd 2003
- Published: 19 November 2003
The recently solved X-ray crystal structures of the ribosome have provided opportunities for studying the molecular basis of translation with a variety of methods including cryo-electron microscopy - where maps give the first glimpses of ribosomal evolution - and fluorescence spectroscopy techniques.
- Functional Center
- Bacterial Ribosome
- Eukaryotic Ribosome
- Cognate tRNA
The bacterial ribosome, a 2.5 MDa assembly formed by three large RNA molecules and 51 proteins, is the most complex biological structure that is currently known at atomic resolution. It is surpassed by some large viruses in sheer number of atoms (250,000 in the ribosome, if we include the hydrogen atoms), but such viruses are typically formed by symmetrically arranged repeats of a unique structure whose size is easily dwarfed by the ribosome, which has no repeated units. The ribosome's function - to translate the genetic code into protein - is one of the most fundamental processes of life, and intense efforts are going into elucidating the underlying mechanisms. These efforts are reminiscent of the heroic struggle to solve another of life's riddles, the question of how genetic information is stored and replicated, which resulted in the discovery of the structure of DNA 50 years ago. A review of research on the ribosome from its discovery in 1957 until the turn of the century is found in Spirin's comprehensive book .
In the ribosome, functional complexity - known from several decades of biochemical studies by thousands of researchers - is matched by structural complexity. Initial visualizations of negatively stained ribosomes by electron microscopy  showed little more than a particle that is subdivided into subunits of unequal size. But a first appreciation of the structural complexity came from cryo-electron microscopy (cryo-EM), which in 1995 started to produce density maps revealing an intricate topology [3, 4] and allowed mapping of the binding positions of critical ligands such as tRNA [5, 6] and elongation factors Tu (EF-Tu ) and G (EF-G ). Subsequently, many years of hard work by several X-ray crystallography groups bore fruit in the year 2000, when three groups published X-ray structures of the small [9, 10] and large  ribosomal subunits of the eubacterium Thermus thermophilus and the archeon Haloarcula marismortui, respectively. The wealth of data that has emerged from these and ensuing X-ray studies of the complete bacterial ribosome , a ribosomal subunit of another species (Deinococcus radiodurans ), as well as numerous complexes of the subunits with antibiotics, has radically changed the direction and pace of research. Even though it has not yet led to an understanding of ribosome function, it has nevertheless provided the essential structural basis required for the elucidation of the underlying molecular mechanisms.
The ribosome is among the most ancient 'inventions' of evolution, and a detailed comparison of ribosome structures encountered in today's species should shed light on the way new functionality in protein synthesis, and the ancillary functions of protein export and control, have developed over the course of 3.5 billion years. Unfortunately, it has been very difficult to grow crystals suitable for X-ray studies from all but a few 'extremophilic' species of bacteria - those that are adapted to growth under extreme conditions of temperature, salt concentration, radiation exposure, or acidity. This restriction has limited our knowledge in two important ways. Firstly, there is still no X-ray structure for the species for which the largest body of data from biochemical, genetic and biophysical experiments is available: Escherichia coli. Secondly, evolutionary comparisons of structures at atomic resolution are currently possible only for a small number of species that are not widely separated in evolution. As yet there is no X-ray structure available for any eukaryotic ribosome, although an atomic model has been built for the ribosome from yeast on the basis of a cryo-EM map and homology modeling . To date, therefore, the bulk of what we can say about the structures of ribosomes from different species and different kingdoms has come from cryo-EM.
The inter-subunit cavity, which is open on both sides, is suitably shaped to act as a conduit for the tRNA's migration through the ribosome from the A to the E site. The 'tRNA entrance' is roughly funnel-shaped and constitutes the binding site for several protein factors: EF-Tu, which delivers the tRNA as part of a ternary complex with GTP to the ribosome; EF-G, which catalyzes the translocation of tRNAs and mRNA by one codon; release factors (RF1, RF2, and RF3), which release the polypeptide in response to the appearance of a stop codon at the A site and prepare the ribosome for recycling; and finally, recycling factor (RRF), which in concert with EF-G separates the subunits from each other and liberates the mRNA and the remaining tRNAs. A prominent feature of the ribosome on the tRNA entrance side is a long, flexible stalk formed by proteins L7 and L12, whose function is still unclear; the base of that stalk is formed by ribosomal protein L10 and a 58-nucleotide region of RNA that is tightly associated with the carboxy-terminal domain of protein L11. This component of the ribosome is instrumental in its GTPase activity, which is required both for EF-G-dependent translocation and accommodation of tRNAs.
The 'tRNA exit,' at the opposite end of the inter-subunit space, is gated by a large mushroom-shaped stalk, the L1 stalk, which is formed from an RNA helix and a globular protein, L1. This stalk is mobile and can pivot along an arc (by at least 30 degrees [13, 21, 22]) around a flex point on the RNA helix. It can apparently alternate between an 'open' position, in which it allows tRNA to exit, and a 'closed' position, in which it blocks the exit (see below).
It has been possible, first by cryo-EM [5, 6, 23] and then by X-ray crystallography [12, 15], to visualize tRNA bound at these sites and to follow the progress of tRNA through the inter-subunit space. As it travels, the tRNA interacts transiently with the two regions of the ribosome called 'functional centers': first the anticodon end interacts with the decoding center, located on the 'neck' region of the small subunit, and second the CCA end interacts with the peptidyl-transferase center, a pocket-like structure on the large subunit made entirely of RNA that also forms the entrance of the polypeptide exit tunnel. The X-ray and cryo-EM maps show many additional contacts of the tRNA with the ribosome in the different stations of its trajectory, suggesting that its movement is tightly controlled for stereochemical precision at the functional binding sites.
After the X-ray structures appeared [9–13], initial attention focused on the molecular processes at the two functional centers. It was suggested that the peptidyl-transferase activity of the ribosome involved a predominantly chemical catalysis of peptide-bond formation , but this hypothesis has given way to the view that the proper positioning of the substrates may be all that is required; this issue is still not resolved . Valuable insights into the decoding process have been gained by the group of Ramakrishnan, who have studied the interaction of a tRNA anticodon loop with the decoding center of the isolated 30S subunit in the presence of mRNA at atomic detail . Decoding and accommodation involve a complex sequence of steps that consist of a dynamic interplay between the ribosome, tRNA, and EF-Tu. Many 'snapshots' will need to be analyzed to get the full picture. Toward this end, cryo-EM is starting to provide the first low-resolution (around 10 Å) density maps of the translating ribosome [26, 27].
In vitro translation systems mimic the conditions in the cell by supplying ribosomal subunits and the basic ligands, as well as GTP, in a carefully balanced buffer solution (see ). Although not as efficient as in the cell, in which protein synthesis proceeds at 20 amino acids per second, protein synthesis in an in vitro system still works at a rate of about 5 amino acids per second, which means that a protein with a length of 200 amino-acid residues can be made in about 40 seconds. Such in vitro systems, in which ribosome complexes are free in solution and unimpeded by crystal contacts, can be used to study their dynamic behavior.
The notion that the ribosome changes its conformation during the elongation cycle goes back to early studies [29–31]. Functional dynamics can be studied by various biophysical techniques, such as cryo-EM, hydroxyl radical probing, fluorescence stopped-flow and quench-flow analysis, and single-molecule fluorescent resonance energy transfer (FRET). These techniques complement one another in giving different aspects of the system's time course and in looking at bulk or individual molecules. Significantly, studies using the different approaches all come to the conclusion that the ribosome undergoes periodic changes in confirmation during the elongation cycle ([32, 33] and J. Puglisi, personal communication).
So far, the most detailed observations have come from cryo-EM. Here, the dynamic behavior is inferred from a series of three-dimensional 'snapshots' that show the ribosome at different stages of the process. Given that cryo-EM visualization is based on the formation of an average over particles with supposedly identical structures, the taking of a meaningful three-dimensional snapshot requires that a large fraction of the ribosomes is trapped in the same state. This can be accomplished by using either antibiotics or non-hydrolyzable GTP analogs. Antibiotics have been likened to wrenches or 'spanners' that are 'thrown into the works'  of the ribosome. They are small molecules that bind at strategic sites and cause the arrest of one or several steps of the dynamic process, mostly by interfering with the required conformational changes. For instance, tetracycline binds to the small subunit at the decoding center and prevents a cognate tRNA from moving into the A site; kirromycin arrests EF-Tu in a conformation that does not allow tRNA accommodation; and fusidic acid prevents a conformational change in EF-G that is required for the factor to leave the ribosome. Nonhydrolyzable GTP analogs, another means of trapping a particular state, bind with high affinity to the GTP-binding site without allowing GTPase activity to take place, resulting in permanent binding of the ligand.
Following such studies, it has been possible, among other results, to observe a large conformational reorganization of the ribosome that occurs in response to the binding of EF-G, termed the ratchet motion [27, 32]. The two subunits rotate relative to each other, and this motion apparently represents the first step in the two-step process of translocation. Early on, Spirin  suggested that the very architecture of ribosomes, as a complex formed by two loosely linked massive subunits, implied the existence of a relative motion between these building blocks. Currently, atomic modeling is being used to study the molecular basis of the conformational reorganization . There is the intriguing possibility, brought out by normal-mode analysis of the ribosome structure (an analysis based on classical mechanics, predicting the most important modes of motion), that the observed motions are closely related to the dynamic properties of the ribosome's gross architecture ( and Y. Wang, A.J. Rader, I. Bahar and R. Jernigan, personal communication).
With regard to the new additions in eukaryotes with apparent impact on the topology, Dube and coworkers  pointed out that for the ribosome of the rabbit reticulocyte, the surface of the 60S subunit interacting with the endoplasmic reticulum membrane (that is, the region surrounding the exit site of the polypeptide) is planar, as though specialized for maximal contact with a planar membrane. This topological trait (see indication in Figure 4c) has been confirmed for all ribosomes of higher eukaryotes investigated so far (yeast [14, 39, 40]; rabbit reticulocyte [38, 40]; and human (C.M.T. Spahn, E. Jan, A. Mulder, R.A. Grassucci, P. Sarnow and J.F., unpublished data)), and it appears to be a universal feature. Regarding additions affecting protein export, we know through Günther Blobel's seminal work (see ) of the existence of a complex apparatus (the 'translocon') for protein insertion into, or export through, the endoplasmic reticulum membrane. The structural implications of this concept are that some of the new ribosomal proteins and RNA additions near the exit site of the polypeptide tunnel must have a role in interacting with the protein exit channel and the signal recognition particle [39, 40, 42]. The additional mechanisms for translational regulation and control that have evolved in eukaryotes are least understood and will be a rich area for structural probing by cryo-EM once an X-ray structure of the eukaryotic ribosome is available. Much more work is clearly needed before a full picture of the eukaryotic ribosome can emerge.
In conclusion, thanks to recent advances in X-ray crystallography and cryo-EM of the ribosome, we now have extensive information on its structure, on the makeup of functional centers, and the beginning of an understanding of its functional dynamics. This picture, however, comes mainly from a few bacterial species. Much less is known about the structural basis of translation in eukaryotes, but first insights have come from cryo-EM of the ribosome of yeast and mammals.
This work was supported by HHMI and grants NIH R37 GM29169 and R01 GM55440. I thank Michael Watters for assistance with the preparation of figures.
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