Like Gaul, protein synthesis divides naturally into three parts: initiation, elongation and termination (Figure ). During initiation, the two subunits of the ribosome are assembled into a complex that has an aminoacylated initiator tRNA and an mRNA bound, ready to make the first peptide bond of a protein [26
]. During termination, a completed protein is released from its tRNA, and the ribosome assembly dismantled so that its components can be recycled [28
]. The steps of the elongation phase, which is the part of the process we will discuss here, constitute a cycle that must be repeated for every peptide bond formed. (A movie showing the three phases of protein synthesis has been created by the Ramakrishnan group at the MRC Laboratory of Molecular Biology and can be accessed from their website [30
].) The purpose of the references provided here, which are entirely to recent articles and reviews, is to give the reader an entrée into the relevant literature, rather than an outline of the history of the field.
Figure 4 The translation cycle. The intiation of translation is complete once an aminoacyl tRNA charged with formylated methionine has been placed in the P site bound to the initiating AUG codon of an mRNA. Elongation begins when a second aminoacyl tRNA recognizes (more ...)
The ribosome catalyzes two chemical reactions: the aminolysis of the ester bonds that link nascent polypeptides to tRNAs during protein synthesis, and the hydrolysis of those same bonds. The amino group used for the aminolysis reaction is the α-amino group of an amino acid that is ester-linked through its α-carboxyl group to the 3' OH of the 3'-terminal nucleotide of a tRNA. The products of this reaction are a peptide that is one residue longer than it was before the reaction, ester-linked to the tRNA that carried the amino acid into the reaction, and a tRNA that has nothing attached to it. Because this reaction transfers a peptide from one tRNA to another, it is referred to as the peptidyl transferase reaction. In the second reaction, which occurs only during the termination phase of protein synthesis, the nucleophile is water, instead of an α-amino group, and peptides are transferred to water (that is, the last tRNA is released from the newly synthesized protein).
Both the peptidyl transferase reaction and the hydrolysis reaction occur at the same site on the ribosome, its peptidyl transferase center (PTC), about which we now know quite a lot. Our understanding can be summarized as follows [31
]. (1) The PTC is located in the center of the subunit interface surface of the large ribosomal subunit. (2) Although RNA-protein interactions are essential for stabilizing the conformation of the PTC, it is composed entirely of RNA: the ribosome is a ribozyme. (3) The catalytic properties of the PTC are not modulated by interactions between the two subunits. (4) The PTC includes a site that accommodates peptidyl tRNAs, the P site, and a site to which aminoacyl tRNAs bind, the A site (see Figure ). (5) Both the A site and the P site of the PTC interact primarily with the 3' terminal CCA sequence that is common to all tRNAs. Thus, to first approximation, differences in tRNA sequences make no difference in the PTC. (6) When the A site of the PTC is empty and its P site is occupied, the PTC adopts a conformation that protects the ester bonds of peptidyl tRNAs from nucleophilic attack. A conformational change accompanies the binding of aminoacyl tRNAs to the A site (as well as the binding of release factors to the ribosome) that exposes the ester bond in the P site to nucleophilic attack. (7) Beyond positioning substrates properly, the PTC seems to do little to enhance the rate of peptide bond formation. (8) The group that makes the largest chemical contribution to the rate of the peptidyl transferase reaction is the 2' OH of the 3'-terminal A of the tRNA in the P site, which facilitates the removal of a proton from the attacking amino group and the addition of a proton to the leaving group, which is the 3' OH of the tRNA bound in the P site. (9) On the ribosome, the peptidyl transferase reaction proceeds at a rate that is about 107
times faster than the rate of similar reactions in solution. (10) Once substrates are bound appropriately to the PTC, the peptidyl transferase reaction occurs at a rate that is at least ten times faster than the overall rate of protein synthesis in living cells, which is about 20 s-1
. (11) At neutral pH, the ester bond of an aminoacyl tRNA is a high energy bond, but the ester bond in a peptidyl tRNA is not. Given that the net effect of the peptidyl transferase reaction is the destruction of a high energy ester bond and the creation of a lower energy peptide bond, the forward direction of the peptide formation is strongly favored thermodynamically.
This description of the peptidyl transferase reaction raises as many questions as it answers. What keeps nascent peptides from inhibiting their own synthesis by filling up the PTC? How are discharged tRNAs removed from the P site? How does the peptidyl tRNA product of the peptidyl transferase reaction move from the A site, where it forms, to the P site, where it must reside if another amino acid is to be added to the nascent peptide chain? How is the next aminoacyl tRNA delivered to the A site?
Product clearance is thought to be the simplest of these issues. As nascent peptides form, they insert into a cavity called the peptide exit tunnel, which extends from the back of the PTC all the way through the body of the large ribosomal subunit [32
]. It is not until the length of nascent peptides exceeds about 40 amino acids that their amino-terminal sequences emerge on the far side of the ribosome and start engaging with the apparatus that ensures protein folding and/or export. As far as we know, nascent poly-peptides diffuse down the tunnel in response to the nudge they are given as each peptide bond forms, but there are hints that it may be more interesting [33
]. Two recent crystal structures have provided insights into how ribosomes carrying completed proteins are recognized and the ester bond linking the protein to tRNAs is hydrolyzed, which is the ultimate step in product clearance [34
The tRNA movements that reset the PTC after each round of peptide bond formation are still only partially understood [36
]. Discussions of this process, which is called translocation, are best begun by reminding the reader that tRNAs are L-shaped RNAs that vary considerably in sequence but are nearly identical in shape. One arm of the L, the acceptor stem, includes the 3' terminal CCA sequence mentioned earlier. The other arm, the anticodon stem, terminates with a loop that includes an anticodon, which is the 3'-nucleotide sequence that pairs with mRNA codons during protein synthesis. (Aminoacyl tRNA synthetases ensure that the amino acids that get esterified to the acceptor stems of tRNAs are the ones encoded by mRNA triplets complementary to the anticodon sequences of those tRNAs.) Messenger RNAs bind to the small ribosomal subunit in the region where its head joins its body (Figure ), and the place on the small subunit where tRNA anticodons interact with mRNA codons is called the decoding center. The A site and the P site of the decoding center are the locations where the anticodons of aminoacyl tRNAs and peptidyl tRNAs, respectively, are bound to the small ribosomal subunit just before peptide transfer occurs. Thus, translocation must reposition tRNAs on both ribosomal subunits. (tRNAs are L-shaped because there is a prominent ridge on the large subunit separating the PTC from the decoding center of the small subunit that only an L-shaped molecule can surmount.)
Translocation on the large subunit precedes translocation on the small subunit [37
], and it seems to be a spontaneous, diffusive process. After peptide bond formation, the acceptor stems of both tRNAs in the PTC move towards the L1 arm of the large ribosomal subunit. The CCA sequence of the discharged tRNA moves from the P site of the PTC to the so-called E (exit) site of the large ribosomal subunit, which can bind only deacylated tRNAs [20
], and the CCA-peptide moiety of the peptidyl tRNA in the A site moves to the P site of the PTC. The A-to-P motion of peptidyl tRNAs is accompanied by a 180° rotation of CCA sequences relative to the bodies of tRNAs. Large-subunit translocation correlates with a rotation of about 10° of the small subunit relative to the large in the direction of the L1 arm, which is called ratcheting. The ratchet motion also seems to result from thermal diffusion, and it is unclear how tightly ratcheting is coupled to large-subunit translocation. Nevertheless, the data suggest that both must occur before small subunit translocation can take place.
Large-subunit translocation leaves the ribosome in a hybrid state, in the sense that the acceptor stem of the peptidyl tRNA is in a P site of the PTC while its anticodon end occupies the A site of the decoding center; the acceptor stem of the discharged tRNA is in the large-subunit E site while its anticodon is in the P site of the decoding center [38
] (Figure ). Small-subunit translocation has two results. First, it advances the ribosome by three nucleotides along the mRNA to which it is bound in the 3' direction, which places a new codon in the A site of the decoding center. Second, it resolves the hybrid state by making the anticodon end of peptidyl tRNA move from the A site to the P site of the decoding center, and the anticodon end of the deacylated tRNA move from the P site of the decoding center to the E site of the small subunit. The anticodon of the tRNA that moves from the A site to the P site of the decoding center remains associated with its codon in the mRNA so that the register in which the mRNA is being translated is maintained. It is unclear whether the anticodons of tRNAs in the E site actually interact with mRNA or not; there are biochemical data indicating they do, but the structural data are ambiguous.
Although small-subunit translocation can occur spontaneously, the spontaneous process is painfully slow. Like all the major steps in protein synthesis, it is catalyzed in the cell by a G protein. The G protein in this case, which is EF-G in bacteria and EF-2 in eukaryotes, binds to the ribosome with a GTP bound that is hydrolyzed in the process [40
]. EM images of EF-G/ribosome complexes, which are all we have, show that this tadpole-shaped molecule binds to the ribosome with its head (which includes its GTPase site) bound to the large ribosomal subunit at the base of the L11 arm. That part of the EF-G binding site includes the sarcin-ricin loop (SRL) of 23S/28S rRNA (Figure ) that, for reasons still unclear, is critically important for the activity of all of the G-protein factors that interact with the ribosome during protein synthesis. The distal end of the tail of the tadpole inserts into the A site of the decoding center of the small subunit. The binding of all proteins that interact with the EF-G binding site is promoted by the L10 complex and the rest of the L11 arm, but the details remain to be worked out.
In solution the GTPase activity of EF-G is very low, but it increases dramatically when the factor binds to the ribosome. Thus, shortly after EF-G·GTP binds to the ribosome, its GTP hydrolyzes. This causes EF-G to undergo a major conformational change that seems to push the anticodon stems of tRNAs across the decoding center, dragging the mRNA with them. Two additional events ensue: the ribosome unratchets; and EF-G·GDP is released into solution. Biochemical data suggest, and EM structures confirm, that the conformational changes that accompany EF-G-assisted translocation are more complicated than this account of translocation seems to require, but until the relevant atomic resolution structures become available, we are unlikely to understand them properly.
Once translocation is complete, the ribosome is ready to bind a new aminoacyl tRNA, and if there is a deacylated tRNA in the E site of the ribosome, aminoacyl tRNA binding is accompanied by release of that tRNA into solution. Biochemical data suggest that these two processes interact with each other, and structural data show that tRNA release correlates with conformational changes in the L1 arm of the large subunit.
Aminoacyl tRNAs are delivered to the ribosome by a second G protein, which is called EF-Tu in bacteria and EF-1α in eukaryotes. The complex that EF-Tu forms with aminoacyl tRNA (and GTP), the so-called ternary complex, resembles EF-G in its overall shape, with the anticodon stem of the ternary complex corresponding to the tail of the EF-G tadpole and its EF-Tu/acceptor stem portion resembling the head. As far as we know, the ternary complex binds to the ribosome the same way that EF-G·GTP does. Its EF-Tu/acceptor stem portion associates with the SRL region of the large subunit and its anticodon stem extends into the A site of the decoding center.
If the anticodon of the tRNA in a ternary complex base-pairs properly with the mRNA sequence in the A site of the decoding center, in other words if the codon and anticodon are cognate, a conformational change occurs that stimulates GTP cleavage and release of EF-Tu·GDP from the ribosome. The aminoacyl tRNA left behind is oriented so that its aminoacyl end is far from the A site of the PTC; the large reorientation required to place its acceptor stem in the PTC is called accommodation. Once accommodation has occurred, the system is ready for the formation of the next peptide bond, which ensues quickly thereafter.