Structural Basis for the Decoding Mechanism
STEVEN T. GREGORY*
[SECTION EDITOR: MICHAEL O’CONNOR]
Posted January 2, 2009
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912
*Mailing address: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912. Phone: (401) 863-3652, Fax: (401) 863-1201, E-mail:
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The accurate translation of genetic information requires the iterative selection of appropriate aminoacyl-tRNA molecules as dictated by mRNA triplets present in the acceptor site of the ribosome. For each codon, the Escherichia coli ribosome must choose, with high fidelity, the correct substrate from among 46 isoacceptor tRNA species having overall structural similarity resulting from their need to interact with common binding sites. Just how the ribosome achieves this discrimination with precision has steadily been uncovered by the combined approaches of genetics, biochemistry, enzymology, and structural biology. The current picture of the decoding process has evolved from the long-term efforts aimed at establishing a complete scheme for the elongation cycle of protein synthesis (for an early review, see reference 81; for a more recent summary, see reference 179). The early pictures of tRNA selection as part of a black-box process have been replaced with high-resolution crystal structures showing conformational transitions and detailed kinetic pathways. It now seems likely that a comprehensive understanding of the decoding process will be established in the not-too-distant future, distilled down to stereochemistry and rate constants. This chapter will attempt to summarize our current state of understanding of the decoding process and present some of the experimental evidence supporting the most current model.
Aminoacyl-tRNAs do not diffuse into the ribosome freely from solution but, rather, are delivered to the ribosome as part of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Upon binding the ribosome, the aminoacyl-tRNA–EF-Tu–GTP ternary complex engages in codon recognition, in which the anticodon bases pair with the three bases of the A site codon. Initial recognition places the ternary complex and ribosome in the so-called A/T state (94), in which the anticodon end of the tRNA is in the A site while the acceptor end remains bound to EF-Tu. Correct codon recognition, in a manner only poorly understood, stimulates EF-Tu to hydrolyze GTP, and the resulting EF-Tu–GDP binary complex disengages from the ribosome. This frees the acceptor end of the tRNA and the esterified amino acid to move into the peptidyltransferase active site, where the aminoacyl-tRNA is positioned to act as the acceptor substrate in peptide bond formation. This movement results in the A/A state (94), in which aminoacyl-tRNA occupies the A site on both 30S and 50S subunits. More recent structural and kinetic studies have created a much more detailed and complete picture of the decoding process (for reviews, see references 118 and 135; for additional interpretations, see reference 105).
Early measurements of the accuracy of decoding led to the realization that experimentally observed error frequencies were substantially lower than could be accounted for by the known thermodynamics of base-pairing specificity alone (reviewed extensively in reference 77). Rather, some mechanism must exist for amplifying the consequences of small differences in cognate and near-cognate binding affinities. An elegant solution to this problem was postulated by Hopfield and Ninio, who proposed the existence of a proofreading step in tRNA selection, whereby the enhancement of accuracy could be achieved by passing tRNAs through two screening steps, the overall accuracy or selectivity of the entire process being the product of the accuracy of the individual steps (64, 104). Thus, tRNAs would undergo an initial recognition, followed by a second, proofreading step. Initial recognition occurs upon the binding of the ternary complex to the ribosome; noncognate aminoacyl-tRNAs are rejected at this point, but near-cognate substrates still bind to a significant extent. The proofreading step in which aminoacyl-tRNA is ejected from the A site occurs subsequent to GTP hydrolysis and the release of EF-Tu–GDP from the aminoacyl-tRNA–ribosome complex.
This model predicts that there should be an excess of GTP hydrolysis when incorrect aminoacyl-tRNA is added to the system (64). Thompson and Stone observed that for noncognate aminoacyl-tRNA, rejection is not accompanied by GTP hydrolysis, indicating that noncognate aminoacyl-tRNA is rejected after initial recognition, but that near-cognate aminoacyl-tRNA rejection is associated with GTP hydrolysis, consistent with the existence of a proofreading step (163). While Thompson and Stone used a partial system with tRNA selection uncoupled from translocation, a subsequent study using a complete poly(U)-dependent system with unfractionated tRNAs also confirmed that excess GTP hydrolysis accompanies missense incorporation (138).
The kinetic proofreading model has been countered with suggestions for allosteric mechanisms for the enhancement of accuracy. Kurland et al. proposed an allosteric model in which, rather than a proofreading step, conformational distortions of tRNA enhance discrimination (76). This model posited that codon recognition induces a conformational change in the anticodon loop, which leads to changes in other parts of the tRNA, such as the TΨC loop. While current crystallographic evidence does not confirm these specific changes, evidence for the contribution of conformational changes in tRNA is beginning to accumulate (74, 159, 169, 170). Nierhaus argued against the occurrence of proofreading and instead proposed that allostery between the A and E sites is sufficient to account for the accuracy of tRNA selection (102). However, the very existence of allostery between the A and E sites has been called into question (149), and at any rate, there is no reason why allostery and proofreading should be considered a priori to be mutually exclusive; proofreading models simply argue for the existence of a second selection step to account for the observed accuracy. In any case, while allostery is likely to play some role in tRNA selection, the preponderance of the evidence clearly favors the existence of proofreading. Results from molecular dynamics simulations of cognate and near-cognate codon-anticodon interactions with the ribosome suggest that the ribosome uses a combination of shape discrimination and hydrogen bond reorganization in some instances and base pair stability in others; also important is the role of the mRNA kink between A site and P site codons in this process (141). In a contrary view, rapid kinetic analysis indicates that the ribosome uses a uniform mechanism for the rejection of near-cognate tRNAs, independent of the stability of base-pairing interactions (54). It is not yet clear how these different views will be reconciled.
One of the most important advances in our understanding of decoding is the dissection of this process into a series of kinetically discrete steps and the precise measurements of elemental rate constants under physiologically relevant conditions. With technological developments, the pre-steady-state kinetics of the entire tRNA selection process has been resolved into a number of kinetically distinguishable steps (Fig. 1) (13, 28, 120, 136). Amino acids are brought to the ribosome esterified at the acceptor end of tRNA as part of a ternary complex of aminoacyl-tRNA, EF-Tu, and GTP. The first step in tRNA selection is the rapid (on the order of 108 M−1 s−1), reversible, initial binding of the ternary complex to the ribosome. This step is thought to first involve interactions between EF-Tu and the ribosomal protein L7-L12 tetramer complex on the 50S subunit (13, 136).
Initial binding is followed by codon recognition, during which the codon-anticodon minihelix forms and which occurs only with cognate and near-cognate complexes. The rate constants for cognate and near-cognate complexes in this step are similar (190 s-1), although the reverse reaction is much faster for near-cognate complexes (80 s-1) than it is for cognate complexes (0.23 s−1). Codon recognition then induces in EF-Tu a conformation that is competent for GTP hydrolysis. This GTPase activation step is much faster for cognate (260 s−1) than for near-cognate (0.4 s−1) tRNA. This coupling of codon recognition and GTP hydrolysis is thought to involve signal transmission from the 30S subunit to the 50S subunit, where the main contacts with EF-Tu take place (125). The precise mechanism by which this signal transmission occurs remains to be determined. This step is followed immediately by rapid GTP hydrolysis, whose rate is limited by the previous step, as is that of a conformational change in EF-Tu to the GDP-bound form. The release of EF-Tu from the ribosome is inhibited by the antibiotic kirromycin, which stabilizes aminoacyl-tRNA–EF-Tu–GDP on the ribosome (123). The GTP- and GDP-bound forms of EF-Tu have both been observed crystallographically and differ dramatically in conformation (72, 106, 153). With the release of EF-Tu from the ribosome, aminoacyl-tRNA undergoes an accommodation step (with a rate constant of 7 s−1 for cognate tRNA) in which the acceptor arm with the esterified amino acid moves into the peptidyltransferase active site, where peptide bond formation occurs in the fastest step of the process. As an alternative to accommodation, aminoacyl-tRNA can be rejected during proofreading. This step is slow for cognate aminoacyl-tRNA (<0.1 s−1), and the rate for near-cognate tRNA is limited by the rate of GTPase activation during initial selection (136). Thus, discrimination between cognate and near-cognate tRNAs is a function of both differential stabilities and the rates of GTP hydrolysis and accommodation (28).
These steps have also been observed using single-molecule fluorescent resonance energy transfer (13). The results of these experiments largely confirm the kinetic schemes previously established by pre-steady-state kinetic experiments and indicate approximately 6-fold-greater selection of cognate over near-cognate tRNA during initial selection and approximately 24-fold-greater selection during proofreading. The observations attained by this approach thus far support a model in which induced fit operates during both initial selection and proofreading (121) and add additional intermediate steps in the kinetic scheme.
In addition to EF-Tu, a number of components of the ribosome itself have been implicated in tRNA selection. Early cross-linking and chemical footprinting experiments identified a number of bases of 16S and 23S rRNA contacted by the ternary complex in the A/T state or by the aminoacyl-tRNA alone in the A/A state. The EF-Tu footprint includes bases G2655 and G2661 in the highly conserved sarcin-ricin loop of 23S rRNA (96), while aminoacyl-tRNA protects primarily residues in 16S rRNA, including G529 and G530 of helix 18 and A1408, A1492, A1493, and G1494 of helix 44 (95). Importantly, the protection of these residues by aminoacyl-tRNA occurs in both the A/T and A/A states.
The binding of the ternary complex to the ribosome has been observed at low resolution by cryo-electron microscopy (cryo-EM) (158, 159, 169, 170). As observed in the later, higher-resolution reconstructions, EF-Tu makes contact primarily with the 50S subunit, specifically, the sarcin-ricin loop of 23S rRNA, ribosomal protein L11 and the 1067 region of 23S rRNA, and the L7-L12 stalk complex. L11 and its associated rRNA binding site, the so-called GTPase center, undergo a conformational change from an open to a closed form. As many as three conformations of the GTPase center are observable by cryo-EM (40). The N-terminal domain of L11 contacts the G domain of EF-Tu, consistent with a role for this contact in GTPase activation. The tRNA moiety contacts rRNA structural elements, including 16S rRNA helices 18 and 44 (containing the decoding site) and 23S rRNA helices 43 (the L11 binding site) and 69 and 95 (the sarcin-ricin loop). Unexpectedly, extensive contact appears to be made between aminoacyl-tRNA and ribosomal protein S12 on the 30S subunit. Helix 69 of 23S rRNA, which forms one of the intersubunit bridges (20, 43, 146, 148), also makes contact with the tRNA in the A/A state after accommodation (187). Of the eight conformational states detected by single-molecule fluorescent resonance energy transfer (13), four have been observed by cryo-EM (40).
An additional tRNA binding state, distinct from the A/T state, has been proposed based on immuno-electron microscopy analysis (150). This intriguing, so-called transorientation hypothesis posits that the anticodon bases in the ternary complex are stacked on the 5' side of the anticodon loop and then undergo a conformational switch to the well-established 3' stacked configuration upon accommodation. However, the transorientation hypothesis was quickly met with skepticism (157), stemming largely from inconsistencies between the predictions of the transorientation model and observations from cryo-EM reconstructions of the ternary complex bound to 70S ribosomes. While cryo-EM reconstructions have thus far utilized the ternary complex stalled in the GDP state by kirromycin (158, 159, 169, 170), there is no evidence to suggest a dramatically different conformation for the GTP form sufficient to warrant a completely new binding state. In addition, there are significant steric clashes between the location of EF-Tu in the transorientation model and the L11-rRNA complex. Furthermore, no direct evidence for a 5' stack has ever been obtained, while every high-resolution structure of tRNA shows the anticodon loop to be in the 3' stack configuration. Since its original inception, no further evidence for the transorientation hypothesis has been forthcoming.
Advances in X-ray crystallography analysis of the ribosome, particularly of the 30S subunit from the extremely thermophilic bacterium Thermus thermophilus, have produced high-resolution images of the ribosome in the process of tRNA selection (reviewed in reference 118). Comparisons of crystal structures of the apoform of the 30S ribosomal subunit with those of 30S subunits in complexes with substrate analogs (anticodon stem-loop [ASL] analogs) reveal a global conformational transition upon codon recognition (117), consistent with the proposed induced-fit model for tRNA selection (121). Upon substrate binding, a series of protein-protein and protein-RNA contacts are formed as others are broken (Fig. 2). On the solvent or back side of the subunit in the apoform, away from the intersubunit space, ribosomal proteins S4 and S5 engage in a series of side chain contacts, primarily ionic in nature. Upon cognate tRNA selection in the A site, on the subunit interface side, contacts between ribosomal protein S12 and 16S rRNA residues are formed. Concurrently, the S4-S5 interface contacts are disrupted. The result is a global closing of the 30S subunit around the substrate. The binding of near-cognate aminoacyl-tRNA induces only partial domain closure. Interestingly, hints of the influence of S4 and S12 on the global conformation of the 30S subunit were detected much earlier by chemical probing of mutant ribosomes (3).
Global conformational changes occurring in vivo in 70S ribosomes are likely to be larger than those observed in the crystal structures of the 30S subunit, which are constrained by crystal contacts (117). This notion is supported by comparison of the open form of the 30S subunit structures to low-resolution (9- and 10-Å) crystal structures of wild-type and streptomycin-dependent mutant 70S ribosomes from E. coli; 70S ribosomes show greater movement of the 30S head than do structures of isolated 30S subunits (174). Nevertheless, the domain closure observed with the 30S subunit alone probably resembles that occurring during both initial selection and proofreading, given that induced fit occurs during both these steps (13, 121). High-resolution structures of the ternary complex bound to the 70S ribosome in the A/T state are eagerly awaited.
The stereochemistry of cognate codon recognition is visible in 3-Å cocrystal structures of the T. thermophilus 30S subunit in a complex with an ASL analog and a U6 hexanucleotide mRNA bound to the A site (116). In addition to the three consecutive Watson-Crick pairs between the ASL and the mRNA, conformational changes in the surrounding 16S rRNA participate in codon recognition (Fig. 3). Two universally conserved bases of 16S rRNA, A1492 and A1493, flip out of helix 44 and make A-minor contacts with the second and first positions, respectively, in the minor groove of the codon-anticodon minihelix. A third 16S rRNA base, G530, switches from the syn configuration in the apoenzyme structure to the anti configuration in the complex and makes contact with the second base of the anticodon and the third base of the codon. The third pair is monitored with fewer geometrical constraints, with C1054 of 16S rRNA making a packing interaction without restricting wobble pair formation. These contacts explain the chemical footprinting pattern observed by Moazed and Noller a decade before the resolution of the cocrystal structures of the T. thermophilus 30S subunit (95).
Through conformational changes in these residues, 16S rRNA directly monitors the stereochemistry of codon-anticodon pairing in a manner consistent with an induced-fit model for substrate recognition (121). This recognition is based on shape complementarity rather than sequence specificity, selecting only canonical Watson-Crick pairing geometry of the codon-anticodon minihelix. Only the first and second codon positions are inspected by these residues, such that the ribosome tolerates wobble pairing at the third codon position, thereby allowing the recognition of all cognate codon-anticodon pairs. These interactions between 16S rRNA and the codon-anticodon minihelix are essential, as mutations of G530 (129) or A1492 or A1493 (185) produce dominant lethal phenotypes in E. coli. Modification interference experiments demonstrated that the methylation of the N-1 of A1492 and A1493 abolishes A site binding (185). The third, or wobble, position is not monitored directly, allowing greater flexibility in the base-pairing configuration at this position. I-C and I-A wobble pairs have been visualized crystallographically in the context of the 30S crystal structure, and as predicted by wobble rules and the previously described crystal structure, the ribosome can accommodate a broader purine-purine base pair at the third position with both bases in the anti configuration and does not require a syn-anti I-A base-pairing configuration (100).
Ultimately, it is these interactions that lead to the global open-closed conformational transition described in the previous section. Given the sensitivity of these large-scale conformational changes and the precise configuration of the codon-anticodon minihelix, it is not surprising that mutations in the ribosome or antibiotics can also have dramatic effects on domain closure and decoding fidelity (see below).
It may be presumed that the ASL-U6 complex in the A site of these 30S structures resembles the codon-anticodon complex as it occurs in the accommodated state, after GTP hydrolysis and the release of EF-Tu–GDP, as would be expected for the binding of free aminoacyl-tRNA from solution (116). One issue that has demanded resolution is the presumed difficulty of codon recognition both in the A/T state prior to accommodation and in the A/A state after the release of EF-Tu. The aminoacyl-tRNA is in distinctly different positions in the two states, and significant movement of the acceptor stem and the rotation of the tRNA must occur upon accommodation and movement into the peptidyltransferase center. One possible solution to this problem is that a significant distortion of tRNA conformation occurs in either the A/T or A/A state. Cryo-EM reconstructions of the kirromycin-stalled ternary complex bound to the 70S ribosome prior to GTP hydrolysis and accommodation, that is, in the A/T state, show a significant distortion of the tRNA such that the codon-anticodon minihelix, the anticodon loop, and the proximal base pairs of the anticodon helix may have identical configurations before and after accommodation (159, 169, 170). While the anticodon loop is fixed relative to the A site codon, the rest of the tRNA molecule is more mobile. These findings have generated renewed interest in tRNA as a more active player in tRNA selection than previously considered.
The idea for the existence of an active role for tRNA in the decoding process has a long history. Concurrent with the proposal of kinetic proofreading in decoding (64, 104), an alternative model for tRNA discrimination proposed that conformational changes in the anticodon loop of tRNA upon codon recognition could amplify the differences in affinity for cognate and near-cognate codons and that these conformational changes would be propagated through the body of the tRNA (76). The potential importance of tRNA conformational dynamics in protein synthesis has been vigorously argued (180).
Experimental evidence of tRNA conformational changes in the selection process comes from several sources. For instance, allosteric conformational changes of tRNA have been invoked to explain the observed increase in the fluorescence of a proflavin label, located at residues 16 or 17 in the D loop, upon initial binding and a further increase upon codon recognition and GTPase activation (120). The unusual Su9 suppressor tRNA mutation, which causes tRNATrp to misread UGG as UGA, is a G24A substitution in the D loop, distant from the codon-anticodon interaction (62). A recent kinetic study found that the G24A mutation increases the forward rate constants for both GTPase activation and accommodation but not the rate constant for rejection (24). Presumably, this mutation accelerates these steps by relieving some structural constraint necessary for discrimination.
A role for tRNA conformational distortion is supported by the intriguing cryo-EM observations of the bending of aminoacyl-tRNA bound to the ribosome in the A/T state (159, 169, 170). As stated earlier, some distortion would be necessary in order for codon-anticodon pairing to occur in the same configuration in both the A/T state, prior to the movement of the acceptor end into the peptidyltransferase center, and the A/A state, after the accommodation and release of EF-Tu–GDP. According to the findings of one cryo-EM study, a dramatic 30° bend in the anticodon stem is observed in the A/T state, which is reduced to about 15° in the A/A state (170). It has been proposed that this distortion of the tRNA in the A/T state may provide the driving force for tRNA movement into the A/A state (40, 117, 169, 170, 184). Such a distortion is consistent with the similar chemical footprints of aminoacyl-tRNA on 16S rRNA in the A/T and A/A states (95). Interestingly, conformational distortions of tRNAs in the P/P and E states (corresponding to the binding states of peptidyl-tRNA in the P site and deacyl-tRNA in the E site) have now also been observed crystallographically in 70S ribosomes from T. thermophilus, and the degree of bending decreases incrementally with each state; thus, a general phenomenon of conformational relaxation may contribute to the progressive movement of tRNAs through the ribosome (74). An all-atom simulation of the accommodation step suggests the importance of the flexibility of the acceptor helix as well as the anticodon helix (142).
One problem that remains to be solved is how the signal that appropriate codon recognition has occurred is transmitted to the large subunit to activate GTP hydrolysis by EF-Tu. Whether this signal is transmitted through the tRNA or through intersubunit bridges connecting the large and small ribosomal subunits, or both, has not been determined. It has been demonstrated that intact tRNA is required to activate GTP hydrolysis; the independent binding of an anticodon-D stem fragment and an acceptor-T stem fragment fails to stimulate GTP hydrolysis, even in the presence of paromomycin, which normally stimulates GTP hydrolysis in the presence of near-cognate tRNA (125). This finding supports the notion that a conformational constraint on the tRNA in the A/T state is required for GTPase activation. An alignment-misalignment hypothesis proposes that the strain induced by the A/T state serves as the signal for inducing GTPase activation and that the misalignment of the tRNA due to near-cognate codon recognition prevents signal propagation (143).
Several types of translational errors occur. Among these are missense errors, in which the ribosome misreads one codon as another. These errors usually involve codons with similar sequences, such as UUU being misread as CUU, coding for leucine rather than phenylalanine. Similarly, nonsense codons can be read as sense codons, as in the case of tRNATrp misreading of UGA as UGG (35) or tRNAGln misreading of UAG and UAA as CAG and CAA, respectively (103). The frequency of such translational errors varies with the mRNA sequence but probably ranges from 5 × 10−5 to 5 × 10−3 events per codon. This topic has been subjected to an extensive review (77) and will not be treated further.
Other types of errors can be lumped together into what can be described as processivity errors. These include events such as drop-off, or the premature release of peptidyl-tRNA from the ribosomal P site; frameshifting, in which the ribosome incorporates aminoacyl-tRNA into the incorrect reading frame; and hopping or bypassing, in which the ribosome ignores a stretch of mRNA sequence. Drop-off, or premature termination, is almost certainly the most deleterious translational error, as it depletes the cell of tRNAs that can be recycled to participate in protein synthesis (92). Nevertheless, drop-off is also the most frequent of translational errors (77). Certain codons, such as AAA, are particularly prone to drop-off, and a specific soluble protein, peptidyl-tRNA hydrolase, encoded by the pth gene, hydrolyzes the acyl bond, thereby freeing the tRNA to return to the translating pool of tRNAs. Cells deficient in this enzyme due to pth temperature-sensitive alleles accumulate peptidyl-tRNA and cease growth at the nonpermissive temperature. The overexpression of tRNALys, which reads AAA and AAG, relieves the growth deficiency caused by the pth mutation, indicating that it is the drop-off of peptidyl-tRNALys at AAA codons and its sequestration that is the primary defect (60). Given the abundance of AAA codons, it seems that even rare drop-off events are detrimental to cell growth.
Frameshifting frequency can be influenced by a number of factors. The intracellular abundance of a particular species of aminoacyl-tRNA can influence frameshifting, such that consecutive rare codons, such as the rare arginine codon AGG in E. coli, can efficiently stimulate frameshifting (155). Monotonous sequences, such as UUUYNN, are also prone to frameshifting (147). Finally, there are programmed frameshifting events that are utilized as a mechanism to control gene expression, and programmed frameshifting events are quite common in viruses. These generally require a slippery, or monotonous, sequence followed by a pseudoknot structure. The structural basis for the requirement of a pseudoknot has been suggested by the findings of a recent cryo-EM study (101).
In the phage T4 gene 60 hop, peptidyl-tRNA in the ribosomal P site disengages from the P site codon, bypasses an astonishing 50 nucleotides of mRNA, and lands at a cognate downstream codon (65). Ribosomes bypassing this 50-nucleotide sequence remain attached to the mRNA and scan to find the downstream landing site (44). Also, mutations in the elbow region of the tRNAGly species that decodes the GGA codons at the takeoff and landing sites have negative effects on bypassing efficiency (59). Mutations in ribosomal protein L9 can also influence this bypass (58), which is curious given the position of L9 on the E site side of the ribosome, distant from the A site, where presumably the takeoff and landing sites are recognized. In the context of the crystal structure of the 70S ribosome, one of the globular domains of ribosomal protein L9 can be seen to partially disengage from the 50S subunit (187) and contact an adjacent ribosome in the unit cell; the biological relevance of this contact is suggested by the fact that it is seen in crystals of ribosomes from both E. coli (146) and T. thermophilus (188). Precisely how such a contact may influence the bypass is yet to be determined.
For some time, it has been known that some ribosome-targeted antibiotics can perturb translational fidelity, with streptomycin being the classic example. Streptomycin was shown to cause phenotypic suppression of the leaky argF40 auxotrophic mutation (46). Thus, in the presence of streptomycin ribosomes would, presumably at some low frequency, read the mutant nonsense codon as sense and incorporate an amino acid, allowing cells to grow in the absence of arginine. Experiments using S30 extracts and ribosomes programmed with synthetic RNA homopolymers showed that aminoglycosides can induce misincorporation in vitro (26). This property of streptomycin is almost certainly the primary basis for the mode of action of this drug and is responsible for its bactericidal effect (27). Other aminoglycosides, such as neomycin and paromomycin (among many others), also cause misreading.
The structural basis for the mode of action of aminoglycosides has been gleaned using a number of genetic and biochemical approaches. That 16S rRNA helix 44 comprises part of the binding site for aminoglycosides of the paromomycin type was first indicated by the identification of aminoglycoside resistance mutations in a number of organisms (30, 82, 154) and by the findings of chemical footprinting experiments (181). That helix 44 is the sole component required for aminoglycoside binding was first demonstrated by chemical footprinting (132) and nuclear magnetic resonance (38) studies using small oligonucleotide minihelices that could bind paromomycin and related antibiotics with high affinity and specificity. Later, cocrystal structures of the entire 30S subunit bound with paromomycin and streptomycin unambiguously identified the binding sites for these drugs (19). To some degree, domain closure that normally occurs upon cognate codon recognition can be artificially induced by the antibiotic paromomycin, and in the presence of both near-cognate aminoacyl-tRNA and paromomycin, domain closure closely resembles that observed with a cognate substrate alone (117). Paromomycin also induces the flipping out of A1492 and A1493, in a manner similar to that observed by the nuclear magnetic resonance analysis of an isolated A site oligonucleotide-paromomycin complex (39). Thus, paromomycin reduces both the energetics of local conformational changes immediately at the site of codon recognition and the energetics of domain closure.
Streptomycin interacts with the 30S subunit almost entirely through contacts with the 16S rRNA backbone via amino and hydroxyl groups of streptomycin and 2' hydroxyls and phosphate oxygens of rRNA; one contact is made between streptomycin and the ε-amino group of K42 of ribosomal protein S12 (19). The rRNA residues contacted by streptomycin are derived primarily from two pseudoknot structures: the so-called central pseudoknot, made up of helices 1 and 27, and the helix 18 pseudoknot. Steptomycin also contacts the backbone of helix 44. Thus, in contrast to paromomycin, whose binding site is contained entirely within helix 44, streptomycin contacts a total of five structural elements of 16S rRNA and ribosomal protein S12.
The results of pre-steady-state kinetic experiments have led to a model in which GTP hydrolysis by EF-Tu is conformationally coupled to codon recognition (53). Such experiments indicate that streptomycin causes an uncoupling of GTP hydrolysis rates from codon recognition. In the absence of antibiotic, the rate of GTP hydrolysis with cognate codon recognition is much greater (250 μM−1 s−1) than that with near-cognate codon recognition (0.4 μM−1 s−1). Streptomycin has the effect of uncoupling codon recognition from the GTP hydrolysis rate, such that the rate of GTP hydrolysis is increased for near-cognate codon recognition and is decreased for cognate codon recognition. The similarity in GTP hydrolysis rates for cognates and near cognates (~2 μM−1 s−1) explains in part the effect of streptomycin on discrimination between cognate and near-cognate tRNAs.
In addition to aminoglycosides, other ribosomally targeted antibiotics have been found to affect translational accuracy. The aminocyclitol kasugamycin, known primarily as an inhibitor of translation initiation, has the opposite effect and decreases errors, in contrast to streptomycin and other aminoglycosides (172). The kasugamycin resistance mutation ksgA, which eliminates the N6,N6-dimethylation of A1518 and A1519 of 16S rRNA (57), produces a ram phenotype (172). Edeine, which binds to the E site of the 30S subunit (128), induces misreading by poly(U)-programmed ribosomes to a degree comparable to the effect of streptomycin (31). Chloramphenicol, a specific inhibitor of the peptidyltransferase activity of the ribosome, causes phenotypic suppression (70) and misreading (162) in vivo despite the great distance of its binding site from the decoding center. Similarly, linezolid causes misreading in vivo (162), even as the genetic evidence (73, 182) places its binding site at or near the peptidyltransferase active site. Just how such drugs affect decoding remains unclear.
Another class of drugs known to influence decoding error frequencies is the tuberactinomycins, which include the structurally related viomycin and capreomycins. These drugs compete with aminoglycosides for binding (93) and induce misreading (89), and mutations conferring resistance to one class of drug often result in cross-resistance to the other (52, 90). Resistance to capreomycin is conferred by the loss of the 2-O-methylation of C1409 in helix 44 of 16S rRNA and C1920 in helix 69 of 23S rRNA of Mycobacterium tuberculosis (67). These data together make a strong case for the binding of tuberactinomycins at the subunit interface. X-ray crystal structures of the 70S ribosome indicate that these two helices interact with each other (14, 20, 148, 187), such that a single molecule of capreomycin may potentially interact simultaneously with both structures (67). Mutations in helix 69 cause misreading (see below).
The first evidence for the role of the ribosome as an active determinant of translational accuracy came from genetic experiments involving antibiotic resistance mutations. The first ribosomal mutations identified were those conferring resistance to the aminoglycoside streptomycin (29). There are four streptomycin phenotypes, including sensitivity (Sms), resistance (Smr), dependence (Smd), and pseudodependence (Smp). Many streptomycin mutations cause ribosomes to be hyperaccurate, as first indicated by their "restriction" of the leakiness of the argF40 auxotrophic mutation (47). This hyperaccuracy is taken to extremes in Smp and Smd mutants, wherein ribosomes require streptomycin to restore tRNA discrimination to tolerable levels. Thus, accuracy is not maximized, but rather there is a tradeoff between speed and accuracy and it is this balance that is perturbed by such mutations. For streptomycin mutants, there is a strong correlation between hyperaccuracy and reduced processivity, with the consequence of a reduced growth rate; normal processivity and growth rates are restored by streptomycin (32, 139).
Mutations affecting the response to streptomycin have most often been identified in rpsL, the gene encoding ribosomal protein S12 (11, 12, 23, 42, 50, 119, 164, 165). Smr alleles of rpsL often express a hyperaccuracy phenotype with reduced levels of misreading, as detected, for instance, by abrogated misincorporation into phage MS2 coat protein (122). Such mutants also have altered kinetic properties (9, 14). Smr, Smp, and Smd mutations in S12 generally cluster in two loops which make extensive contact with 16S rRNA and form part of the streptomycin binding site (Fig. 4) (19). In one of the most comprehensive screens reported (165), the mutations K42R, K42N, K42I, K42Q, and K87R were found to confer an Smr phenotype and P41L, K43E, ΔK87, P90R, P90L, G91D, and ΔR93 were determined to confer Smd phenotypes in E. coli. Mutants expressing a similar spectrum of mutations were analyzed for in vitro protein synthesis activity and the propensity to generate missense errors (23). Such rpsL mutant alleles have been identified in a wide range of species, including the thermophile T. thermophilus (50), Helicobacter pylori (166), and Borrelia burgdorferi (25), among others. Further, the evolutionary conservation of the accuracy effects of such mutations has been demonstrated in the eukaryote Saccharomyces cerevisiae (5). The K42T, K42Q, and K42N mutations in the yeast ribosomal protein S12 homolog, S28, produce hyperaccuracy effects similar to those produced by the bacterial mutations and result in interaction with suppressor alleles for the ribosomal protein S4 and S5 homologs. However, the K42R mutation appears to decrease accuracy in yeast, while it has little effect on accuracy in bacteria.
Smr mutations in 16S rRNA occur in the central pseudoknot comprising helix 1 and the base of helix 27 (45, 55, 78, 98, 127), or in the pseudoknot within helix 18 (91, 156). Thus far, a single 16S rRNA Smd mutation has been identified, the insertion of a C after residue C522 in helix 18 (63). Such rRNA mutations do not arise in genetic selections with E. coli, perhaps owing to the presence of multiple rrn operons combined with the recessive nature of streptomycin resistance (79). Instead, putative Smr mutations have been introduced by site-directed mutagenesis into plasmid-borne E. coli rrn operons (41, 78, 91, 127, 130), although the resistance of the resulting mutants to streptomycin must be ascertained in vitro (78, 91, 127) or in vivo in the presence of wild-type ribosomes which are temporarily functionally inactivated (130). It is, of course, now possible to obtain or construct Smr mutations in rRNA genes in E. coli by using the delta 7 prrn strains in which all seven chromosomal rrn operons have been deleted and rRNA is derived from a multicopy plasmid (6, 7).
Smr mutations in 16S rRNA were initially identified in chloroplasts, which possess single rrn operons (45, 55, 98), and more recently in a species of Mycobacterium having a single rrn operon (37) and in a Mycobacterium smegmatis strain engineered to contain a single rrn operon (156). Streptomycin resistance has also been attributed to an A1408G mutation in helix 44 of T. thermophilus; this mutation was originally selected for resistance to kanamycin and was later found to confer streptomycin resistance when present in both of the two 16S rRNA gene copies found in this species (51). Unfortunately, little effort has been exerted to establish the effects of 16S rRNA Smr mutations on translational accuracy, although the C912G mutation in E. coli was found to produce a hyperaccuracy phenotype in vivo (86). Mutations in helix 27 of yeast ribosomes also affect accuracy, although the phenotypes associated with these mutations do not precisely mirror those associated with their bacterial counterparts (173).
High-resolution crystal structures of the T. thermophilus 30S ribosomal subunit bound with streptomycin (19) allow some predictions to be made as to the mechanism of streptomycin dependence and its suppression and the effects of Smr mutations on translational accuracy (117). As described above, the 30S subunit is seen to undergo a conformational transition from an open to a closed form upon cognate codon recognition. Mutations shifting the conformational equilibrium toward the open form are predicted to cause hyperaccuracy, while those favoring the closed form are predicted to create an error-prone phenotype. Thus, some hyperaccuracy alleles of rpsL abolish specific contacts made between S12 and 16S rRNA upon domain closure. For instance, R53L, the S12 mutation yielding the highest level of hyperaccuracy among all such mutations ever described (167), disrupts a salt bridge between the mutated residue and the ribose-phosphate backbone of helix 44 of 16S rRNA that forms upon domain closure. The resistance mechanism is distinct from many others, as it does not involve the loss of contact with streptomycin, R53 (K53) being some distance from the streptomycin binding site (19). In contrast, the least restrictive Smr mutation is K42R. K42 makes direct contact with both 16S rRNA and streptomycin, and replacement with arginine breaks the contact with streptomycin but not the contact with 16S rRNA, thus not affecting conformational dynamics to any significant extent (19). Other substitutions at position 42 exhibit various degrees of restrictiveness (23).
The precise mechanism of streptomycin dependence is not as yet understood, despite enormous amounts of structural data and numerous genetic studies. Results from chemical probing experiments show that Smd mutations influence the conformation of G530, in a manner opposite that of streptomycin and neomycin (131). High-resolution crystal structures of a T. thermophilus 30S subunit with a P90L Smd substitution in S12 reveal no detectable conformational change (18), suggesting the effects of this mutation to be manifested in kinetic terms inaccessible to a crystallographic approach. Suppressors of streptomycin dependence are readily isolated. Such Smi mutations were originally identified by Rosset and Gorini (137), who described them as ram mutations, for ribosomal ambiguity, due to the associated error-prone phenotypes when these mutations are separated from the original Smd allele. The ram mutations are most often found to occur as alleles of rpsD and rpsE, encoding ribosomal proteins S4 (137, 171, 189) and S5 (126), respectively, although a 16S rRNA ram allele has been described (4).
The structural basis for suppression by rpsD and rpsE alleles can be inferred from the 30S subunit structures; the sites of amino acid substitutions in ram mutants are at the interface between S4 and S5, thus destabilizing the open conformation and favoring domain closure. Mutations favoring domain closure would be predicted to enhance the binding of all tRNAs, including near-cognate ones, in much the same way that error-inducing drugs enhance near-cognate tRNA binding (19, 117). There is not, however, a simple compensatory relationship between S4-S5 and S12 mutations, as not all rpsD mutants exhibit ram phenotypes. The Salmonella enterica serovar Typhimurium Q53L, ΔV200, and 201UGA rpsD alleles in fact cause hyperaccuracy and, perhaps even more remarkably, confer appreciable levels of streptomycin resistance (12). One possible interpretation, though highly speculative, is that these particular rpsD alleles affect domain closure in a manner opposite to that of ram alleles, by stabilizing the S4-S5 interface interaction. Further, mutations at position 42 of S12 act as intragenic suppressors of the Smd mutations at position 90 of the same protein; thus, K42R suppresses P90L in Salmonella (12) and K42T suppresses P90R in T. thermophilus (16). Neither of these mutations produces a ram phenotype; the K42R allele is a nonrestrictive Smr allele (168), while the K42T allele in Salmonella is restrictive (12). Thus, a simple picture of the relationship between these proteins and their phenotypes is neither readily apparent nor subject to a simplistic interpretation.
Recently, a novel rpsE mutant was isolated by selecting for spectinomycin resistance and subsequently screening for cold sensitivity (71). This mutant exhibits a subunit assembly defect, and the mutation also causes increased misreading and frameshifting in vivo. The identified substitution, G28D, is positioned near previously identified rpsE spectinomycin resistance mutations but not near ram mutations, suggesting a distinct mechanism not directly related to the open-closed transition. The nature of this mechanism is difficult to ascertain, given the assembly defect.
Finally, a mutation in the large subunit, the eryB mutation altering ribosomal protein L4, has been shown to have various effects on translational fidelity (113). A number of mutations affecting the large subunit, particularly those leading to alterations of 23S rRNA, are now known to influence decoding (see below).
Given the highly cooperative nature of protein synthetic machinery, it should come as no surprise that mutations affecting the fidelity of decoding are not limited to the 30S subunit. Correct codon recognition results in fast hydrolysis of GTP and the release of EF-Tu–GDP from the ribosome, and mutations in EF-Tu that perturb the rate of GTP hydrolysis are likely to affect decoding accuracy. The antibiotic kirromycin, which binds to EF-Tu, allows GTP hydrolysis but prevents a conformational change in EF-Tu that is required for the factor’s release from the ribosome (123). Mutations in the tuf genes encoding EF-Tu can confer kirromycin resistance. However, there are two tuf genes in most bacteria, including E. coli and Salmonella, and kirromycin resistance is recessive. Thus, kirromycin resistance requires either that both genes are mutant forms or that the wild-type sensitive gene is inactivated. Such mutations have been found to produce error-prone phenotypes and in fact have been identified in genetic selections for suppressors of streptomycin dependence (168, 190). One of these mutant forms is the classic tufAr allele, incorporating an A375T substitution (190), which is identical to the tufA8 mutant allele identified in Salmonella (167). This mutation confers dominant kirromycin resistance in the presence of the highly restrictive rpsL500 Smr allele, incorporating the aforementioned S12 R53L substitution. In fact, it was in the kirromycin-sensitive, tufAr background that the rpsL500 allele was selected for kirromycin resistance. Not only does tufAr suppress streptomycin dependence but it also abolishes the Smr phenotype of rpsL500. This suggests that rpsL500 confers resistance by a mechanism distinct from those associated with other S12 mutations and instead influences the precise manner in which the ternary complex interacts with the ribosome, as well as the effect of streptomycin on domain closure.
Mutations affecting accuracy have also been identified in rRNA genes by using selection for suppressors of streptomycin dependence or suppressors of nonsense or frameshift mutations in reporter genes (Fig. 5 and 6). One of the first of such examples is a U1469C mutation in helix 44 of 16S rRNA, selected as a suppressor of an Smd allele of rpsL (4). This mutation on its own increased the misincorporation of leucine into polyphenylalanine by poly(U)-programmed ribosomes in vitro, at levels comparable to those of S4 and S5 ram mutations. U1469 is distant from the decoding center, and it is not immediately obvious how the mutation of this residue exerts its effect. More recently, the 16S rRNA mutation C912U was identified in a similar process of selection for suppressors of the Smd phenotype conferred by the P90R substitution in ribosomal protein S12 (175). The suppressor phenotype is temperature sensitive, and not all Smd alleles of rpsL are suppressed. The reader will recall that the C912U mutation was first identified as a Smr mutation in chloroplast rRNA genes (45, 55, 98). It is also worth reiterating the observation that C912U, like other Smr mutations, is error restrictive yet suppresses streptomycin dependence, indicating that the capacity to enhance translational error frequencies does not correlate with the suppression of the dependence phenotype.
Genetic selection for suppressors of a UGA nonsense mutation led to a model for nonsense codon recognition by 16S rRNA via base pairing. The deletion of C1054 in helix 34 of E. coli 16S rRNA suppresses a UGA nonsense mutation at position 211 in trpA (99) (Fig. 5). It was hypothesized that the suppressor phenotype of this mutation results from the disruption of a base-pairing interaction between UGA termination codons and a UCA triplet (positions 1199 to 1201) in helix 34, across the helix from C1054, and that this codon recognition occurs during the normal termination step of protein synthesis. Using a similar process of genetic selection for suppressors of a UGA nonsense mutation at position 243 of trpA produced the C1054U mutation and also the C1200U mutation (49). However, results of further mutagenesis studies and quantitative measurements of suppression efficiencies indicate that mutations of C1054 do not act in a codon-specific manner but instead produce a generalized ram phenotype, causing the suppression of all three nonsense codons as well as stimulating frameshifting (97). The apparent specificity for UGA suppression may simply be the consequence of the inherently greater leakiness of UGA nonsense mutations than of UAG and UAA nonsense mutations (35, 103).
An explanation of the phenotypes associated with mutations at position 1054 and in the immediate vicinity is suggested by the location of the C1054 residue in the structures of the 30S subunit (116) and the 70S ribosome (148, 187). C1054 stacks on G34 of the anticodon of A site tRNA, such that mutations of C1054 may be expected to alter the stability or orientation of the ASL. However, these structures do not support direct base pairing between helix 34 and A site codons, as hypothesized in the original model, although given the contact of release factors (RFs) with termination codons in the A site (124), an influence of mutations of C1054 on RF function remains plausible. Mutations disrupting tertiary contacts involving helix 34 also affect decoding accuracy and frameshifting (75).
Substitutions for G517 (108) or G529 (144) in helix 18 produce a generalized ram phenotype (Fig. 5). Not surprisingly, the G1491A mutation in the C1409-G1491 base pair in helix 44 around the so-called decoding site also causes a generalized ram phenotype (49), while a paromomycin resistance mutation at position 1409 has long been known to cause frameshifting in yeast (176). This part of helix 44 makes contacts with ribosomal protein S12 during domain closure, and the C1409-G1491 base pair is adjacent to the two adenosines, A1492 and A1493, that flip out during codon recognition (116). In one study, mutations at a number of sites within 16S rRNA were shown to perturb decoding accuracy and frameshifting (112). These were located at positions suggested in a variety of previous studies to be important for function, and while these sites appear to be dispersed throughout the 16S rRNA secondary-structure map (Fig. 5), they nevertheless are in close proximity when examined in the context of the three-dimensional structure of the molecule, and high-resolution crystal structures place all of these sites near the site of codon recognition.
Given the cooperativity of both subunits in interaction with tRNA and EF-Tu, it should be no surprise that mutations affecting decoding fidelity have been found on the large subunit as well (Fig. 6). The first of these to be isolated was a G1093A substitution in the so-called GTPase center, which forms the binding site for ribosomal protein L11 and interacts, together with L11, with EF-Tu (96, 159, 169) and EF-G (1). The mutant allele was isolated twice independently as a suppressor of UGA nonsense mutations at trpA codons 211 (66) and 243 (49). As in the case of the deletion of C1054 of 16S rRNA, the G1093A mutation appeared to be specific for the readthrough of UGA over UAG and UAA (66). Subsequent site-directed mutagenesis analyses of the two 23S rRNA loops that constitute the GTPase center identified three additional residues, A1067, U1094, and A1095, the mutation of which produces UGA-specific readthrough, leading to the conclusion that these residues contact the termination factor RF-2 (183). A recent high-resolution crystal structure of the 70S ribosome in a complex with RF-1 and RF-2 (124) demonstrated the interaction of both RFs with the GTPase center in the vicinity of the UGA suppressor mutations. However, the basis for the UGA specificity observed in the genetic studies is not clear from the crystal structure, as conformational changes induced in both factors are observed. Given the well-established interaction of the GTPase center with EF-Tu (96, 159, 169, 170), it seems highly unlikely that all of these mutations would have no effect on decoding and be limited to UGA-specific termination.
Another site of interaction with EF-Tu has also been implicated in decoding accuracy. The so-called sarcin-ricin loop, or helix 95 of 23S rRNA, is cleaved by α-sarcin at residue G2661 (Fig. 6) (56, 145). A2660 is depurinated by the plant cytotoxin ricin (88). In either case, the binding of EF-Tu is abolished (17, 56). The sarcin-ricin loop was also shown by chemical footprinting to interact with EF-Tu (96), and the direct contact of the ternary complex with this loop has also been observed by cryo-EM (159, 169, 170). The G2661C mutation in the sarcin-ricin loop of E. coli ribosomes reduces the binding of cognate ternary complex to the ribosome and, when combined with restrictive Smr alleles of rpsL, produces an Smd phenotype (10). This effect could be reversed by a third mutation affecting EF-Tu (160). Genetic selection for suppressors of the trpE91 frameshift mutation gave rise to the C2666U mutation (111). This mutation causes effects on the readthrough of all three stop codons and frameshifting in both directions. A mutation in the yeast sarcin-ricin loop also causes translational errors (85). This mutation alters the closing C-G base pair with a U-G wobble pair, potentially perturbing the conformation of the GAGA tetraloop. A number of mutations in the sarcin-ricin loop are lethal, consistent with a critical function in ribosome-factor interactions (21, 22, 87). A specific tertiary interaction between A2662 in the sarcin-ricin loop and A2531 in the peptidyltransferase center may be important for conformational signaling between the two functional centers, as mutations A2662C and A2531G individually produce dominant lethal phenotypes but give rise to viable cells when combined (21).
Mutations affecting the accuracy of decoding have also been identified in and around the peptidyltransferase active site (Fig. 6). U2504C in yeast mitochondrial 21S rRNA restricts frameshifting (177). A mutation of U2555 in this loop suppresses the trpE91 frameshift mutation (109). G2553, in the A loop, pairs with C75 of the CCA end of A site-bound aminoacyl-tRNA (69). The mutation of U2555 to either A or G, but not C, causes increased readthrough of all three stop codons and both +1 and −1 frameshifting. Similarly, mutations in the P loop, which pairs with the CCA 3' terminus of P site-bound tRNA (107, 140), cause increased readthrough of stop codons and increased frameshifting (48), perhaps by decreasing the binding affinity of P site tRNA and concomitantly increasing A site binding affinity, as observed for ribosomal protein mutations in the 30S subunit (68). More recent data suggest that mutations of G2252 cause stop codon readthrough by inhibiting RF-dependent peptide release activity (36). The mutations G2447C and G2447A, as well as all three base substitutions for the universally conserved A2451, increase misreading and frameshifting (161). Mutations of A2451 have also been shown to affect peptide release (186). Finally, mutations in helix 89 have also been detected in selections for trpE91 suppressors (110).
Finally, we consider accuracy mutations in the intersubunit bridges connecting the 30S and 50S subunits. The first such mutations were identified as trpE91 suppressors located in helix 69 of 23S rRNA (110), and other mutations in this helix have since been shown to increase frameshifting and nonsense readthrough, in addition to producing subunit association defects (61). It is now recognized from cryo-EM analysis (43) and crystal structures (20, 74, 146, 148, 187) of the 70S ribosome that helix 69 participates in an intersubunit-bridging interaction contacting 16S rRNA helix 44 near the decoding site. Further, helix 69 contacts the acceptor helix of aminoacyl-tRNA bound in the A/T state (159, 170) and is suspected of participating in the deformation of the tRNA during tRNA selection (40). As mentioned earlier, antibiotics of the tuberactinomycin family interact with both helix 44 of 16S rRNA and helix 69 of 23S rRNA and induce misreading (89). The deletion of helix 69 produces a dominant lethal phenotype with the predictable subunit association defect, although such ribosomes are functional in vitro with, surprisingly, no apparent defects in accuracy (2). This drastic mutation does produce a defect in RF-1-mediated termination, consistent with the termination-specific effects resulting from the loss of the pseudouridylation of helix 69 (34). Nevertheless, the lethal Δ1916 mutation in helix 69 has also been shown to increase missense errors, suggesting that alterations of this helix can impact either or both termination and decoding, perhaps depending on the nature of the alteration (115). The lethality of the Δ1916 mutation may be suppressed by either of two base substitutions in 16S rRNA, G1048A and U1471C, located in the head and helix 44, respectively (115). G1048 is positioned such that it may conceivably influence domain closure of the head of the 30S subunit during codon recognition. U1471 is adjacent to the original U1469C Smi mutation (3), discussed above, and both residues are close to one of the intersubunit bridges. The observation that mutations in one intersubunit bridge can influence the phenotype of a mutation in another bridge is indicative of the level of cooperativity of the network of intersubunit-bridging interactions in the decoding process. A systematic study of the effects of mutations in several intersubunit bridges indicates that many of these interactions contribute to the accuracy of tRNA selection (84).
While the sheer number of mutations affecting the accuracy of decoding is impressive, it remains to be established which step of the decoding process is affected by each of these changes. In the absence of a systematic, pre-steady-state kinetic analysis of such mutations, it is not at all clear whether a given mutation affects initial recognition, proofreading, or both.
Among the earliest mutations known to suppress nonsense and frameshift mutations were those affecting tRNA. Those most commonly identified are the mutations that modify codon recognition by alterations in and around the anticodon. Many of these alter decoding in a predictable manner via base substitutions in the anticodon itself. We will not concern ourselves with suppressor mutations in the anticodon, as they have been treated extensively elsewhere (33). Instead, we briefly consider other unusual tRNA suppressors that act in ways that are not readily apparent upon inspection of their locations in the structure of the tRNA molecule. The iconic suppressor of this type is the Hirsh suppressor mutant form of tRNATrp, which has acquired a G24A substitution in the D arm, allowing it to read UGA in addition to the lone tryptophan codon UGG (62). A recent kinetic analysis indicates that this mutation causes an acceleration of both initial selection and GTP hydrolysis (24). Exactly how it does so is unclear, as no detailed information regarding the structural consequences of the G24A mutation currently exist. A crystal structure of the Hirsh suppressor tRNATrp, either on its own, as part of the ternary complex, or bound to the ribosome, may be of some help in this regard. It is perhaps noteworthy that the G24A mutation replaces a U11-G24 wobble pair with a Watson-Crick U11-A24 pair which, curiously, is phylogenetically the most frequent base pair at this position in tRNATrp (data were obtained from the comparative RNA database; also see reference 15). A C-G base pair is almost as frequent, while the U11-G24 wobble pair is in fact quite rare, being found in only a small fraction of tRNATrp sequences. This suggests that some other, higher-order structural effect is at work.
Other examples of unusual suppressors include frameshift suppressor mutations in tRNAPro (133) and in tRNAVal (114) and tRNAArg (80). The tRNAPro mutations include a number of mutations in the acceptor stem, D stem, and anticodon stem. Three of these replace Watson-Crick C-G pairs with U-G wobble pairs within helices, while four replace C-G pairs with C-A wobble pairs; insertions in the anticodon loop and a deletion in the variable loop were also found. Mutations in tRNAVal acting as suppressors of the trpE91 frameshift mutation include, in addition to insertions in the anticodon loop, the mutations G39A and G42A in the anticodon helix and the deletion of the variable-loop residue G45, involved in a base triple interaction (114). The Salmonella sufF44 mutation is a C61U transition in tRNAArg, encoded by argU (80). Other argU alleles include those incorporating G53A, at the base which pairs with C61, and C56U in the TΨC loop. They appear to induce frameshifting by decreasing the aminoacylation or stability of tRNA produced by argU, thereby leading to frameshifting by tRNAGlu at the adjacent codon.
While it is not clear how such mutations, which undoubtedly distort the three-dimensional structure of the tRNA molecule, exert their phenotypes, it is tempting to speculate that the bending of the anticodon stem observed in cryo-EM studies (169, 170) is relevant. It is quite possible that in some cases, mutations exist which facilitate such bending so as to increase the forward reaction rates. A more complete picture of the structural effects of these mutations may result from high-resolution structural studies of mutant tRNAs. What such observations do suggest is that a renewed interest in tRNAs as active participants in tRNA selection is warranted.
While information regarding the role of postsynthesis modifications of tRNAs in translational fidelity (see Chapter Transfer RNA Modification), much less is known regarding the impact of such modifications of rRNAs. Even so, it is clear that rRNA modifications have a wide range of impacts on ribosome function (for a review of rRNA modifications, see Chapter Modified Nucleosides of Escherichia coli Ribosomal RNA).
The absence of N6,N6-dimethylation of 16S rRNA residues A1518 and A1519 in ksgA mutants causes, in addition to resistance to the antibiotic kasugamycin, increased misreading in both A and P sites (112, 172). The loss of the ribose methylation of U2552 in the A loop of 23S rRNA, by virtue of mutant alleles of rrmJ, causes the restriction of nonsense readthrough and frameshifting (178), an effect opposite to that of mutations at U2555 in the same loop (109). The loss of modification of residues in helix 69 of 23S rRNA also perturb translational accuracy in yeast (83), consistent with the identification of error-inducing mutations in this helix (see above). However, the loss of pseudouridylation of helix 69 in E. coli leads to termination defects but does not affect miscoding (34). Of some interest, and which have yet to be resolved to any great extent, are the effect of rRNA mutations on modification and the degree to which the phenotypes conferred by such mutations are the indirect result of the undermodification of nearby residues.
One interesting phenomenon is a connection between translational miscoding and mutagenesis. Mutator alleles of tRNAGly, mutA and mutC, were identified and predicted to misincorporate glycine in place of aspartic acid, and it was proposed that the mutator phenotype results from the production of error-prone molecules of DNA polymerase subunits, such as the ε-subunit of DNA polymerase III, encoded by dnaQ, also known as mutD (151). This model is supported by the observation that synthetic mutD alleles can be produced by specific aspartic-acid-to-glycine substitutions (152).
These observations suggest that other mechanisms for inducing translational errors may also produce mutator phenotypes. Indeed, Smr mutants display a mutator phenotype when grown in the presence of streptomycin (134), and the ram mutants bearing the rpsD14 allele are also mutators (8). There probably exist a number of physiological consequences of translational errors that have yet to be discovered.
The time may be rapidly approaching when a complete picture of the tRNA selection process is at hand. Most, if not all, of the steps in the process have been observed and dissected kinetically, and the structural basis of codon-anticodon recognition by the ribosome has been modeled at the level of atomic resolution. Nevertheless, major issues remain unresolved. For instance, the mechanism by which cognate codon recognition on the 30S subunit stimulates GTP hydrolysis by EF-Tu is largely unknown, as is the role of the numerous contacts between the ternary complex and the ribosome in transmitting this signal. Conformational distortions of aminoacyl-tRNA in the A/T state have been observed in cryo-EM reconstructions, but once again, the structural changes and their role in decoding are not known with any precision. Both these questions, if they are likely to be answered, will be resolved by a combination of single-molecule studies, the determination of crystal structures of the ternary complex bound to the 70S ribosome, and genetic experiments directly testing models suggested by X-ray crystallography or cryo-EM.
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Module2.5.4
