Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.     

Significance of the third tRNA binding site, the E site, on E. coli ribosomes for the accuracy of translation: an occupied E site prevents the binding of non-cognate aminoacyl-tRNA to the A site.

The E site (exit site for deacyl-tRNA) has been shown to be allosterically linked to the A site (aminoacyl-tRNA binding site), in that occupation of the E site reduces the affinity of the A site, and vice versa, whereas the intervening peptidyl-tRNA binding site (P site) keeps its high affinity. Here the question is analysed of whether or not the low affinity state of the A site caused by an occupied E site is of importance for the ribosomal accuracy of the aminoacyl-tRNA selection. In a poly(U) dependent system with high accuracy in poly(Phe) synthesis, the acceptance of the cognate ternary complex Phe-tRNA--EF-Tu--GTP (which has the correct anticodon with respect to the codon at the A site) was compared with the competing acceptance of ternary complexes with near-cognate Leu-tRNA(Leu) (which has a similar anticodon) or non-cognate Asp-tRNA(Asp) (which has a dissimilar anticodon), by monitoring the formation of AcPhePhe, AcPheLeu or AcPheAsp, respectively. Cognate (but not near-cognate) occupation of the E site reduced synthesis of the 'wrong' dipeptide AcPheLeu only marginally relative to that of the cognate AcPhe2, whereas the formation of AcPheAsp was decreased as much as 14-fold, thereby reducing it to the background level. It follows that the allosteric interplay between E and A sites, i.e. the low affinity of the A site induced by the occupation of the E site, excludes the interference of non-cognate complexes in the decoding process and thus reduces the number of aminoacyl-tRNA species competing for A site binding by an order of magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

The presence of pseudouridine in the anticodon alters the genetic code: a possible mechanism for assignment of the AAA lysine codon as asparagine in echinoderm mitochondria

It has been inferred from DNA sequence analyses that in echinoderm mitochondria not only the usual asparagine codons AAU and AAC, but also the usual lysine codon AAA, are translated as asparagine by a single mitochondrial (mt) tRNAAsn with the anticodon GUU. Nucleotide sequencing of starfish mt tRNAAsn revealed that the anticodon is GPsiU, U35 at the anticodon second position being modified to pseudouridine (Psi). In contrast, mt tRNALys, corresponding to another lysine codon, AAG, has the anticodon CUU. mt tRNAs possessing anti-codons closely related to that of tRNAAsn, but responsible for decoding only two codons each-tRNAHis, tRNAAsp and tRNATyr-were found to possess unmodified U35 in all cases, suggesting the importance of Psi35 for decoding the three codons. Therefore, the decoding capabilities of two synthetic Escherichia coli tRNAAla variants with the anticodon GPsiU or GUU were examined using an E.coli in vitro translation system. Both tRNAs could translate not only AAC and AAU with similar efficiency, but also AAA with an efficiency that was approximately 2-fold higher in the case of tRNAAlaGPsiU than tRNAAlaGUU. These findings imply that Psi35 of echinoderm mt tRNAAsn actually serves to decode the unusual asparagine codon AAA, resulting in the alteration of the genetic code in echinoderm mitochondria.     

Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.     

On the role of the starved codon and the takeoff site in ribosome bypassing in Escherichia coli

Translating ribosomes can skip over stretches of messenger RNA and resume protein chain elongation after a "bypassed" region. We have previously shown that limitation for isoleucyl-tRNA can initiate a ribosome bypass when an AUA codon is in the ribosomal A-site. We have now generalized this effect to other "hungry" codons calling for four different limiting aminoacyl-tRNA species, suggesting that a pause at any A-site will have this effect. We have assessed bypassing in a large family of reporters with nearly every different triplet in the "takeoff site", i.e. the P-site on the 5' side of the hungry codon, and an identical "landing site" codon 16 nucleotides downstream. The different takeoff sites vary over a factor of 50 in bypassing proficiency. At least part of this variation appears to reflect stability of the codon Colon, two colons anticodon interaction at the takeoff site, as indicated by the following: (a) the bypassing proficiency of different tRNAs shows a rough correlation with the frequency of A Colon, two colons U as opposed to G Colon, two colons C pairs in the codon Colon, two colons anticodon association; (b) specific tRNAs bypass more frequently from codons ending in U than from their synonym ending in C; (c) an arginine tRNA with Inosine in the wobble position which reads CGU, CGC, and CGA bypasses much more frequently from the last codon than the first two synonyms.     

Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex

The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA) in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here we report the 13 A resolution three-dimensional reconstruction determined by cryo-electron microscopy of the kirromycin-stalled codon-recognition complex. The structure of the ternary complex is distorted by binding of the tRNA anticodon arm in the decoding center. The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1 of EF-Tu near the nucleotide-binding pocket. These results provide a detailed snapshot view of an important functional state of the ribosome and suggest mechanisms of decoding and GTPase activation.     

Species-specific codon context rules unveil non-neutrality effects of synonymous mutations

BackgroundCodon pair usage (codon context) is a species specific gene primary structure feature whose evolutionary and functional roles are poorly understood. The data available show that codon-context has direct impact on both translation accuracy and efficiency, but one does not yet understand how it affects these two translation variables or whether context biases shape gene evolution.ConclusionsSince in vivo studies provide evidence for a role of codon context on decoding fidelity in E. coli and for decoding efficiency in mammalian cells, our data support the hypothesis that, like codon usage, codon context modulates the evolution of gene primary structure and fine tunes the structure of open reading frames for high genome translational fidelity and efficiency in the 3 domains of life.     

Steric Complementarity in the Decoding Center Is Important for tRNA Selection by the Ribosome

Accurate tRNA selection by the ribosome is essential for the synthesis of functional proteins. Previous structural studies indicated that the ribosome distinguishes between cognate and near-cognate tRNAs by monitoring the geometry of the codon–anticodon helix in the decoding center using the universally conserved 16S ribosomal RNA bases G530, A1492 and A1493. These bases form hydrogen bonds with the 2′-hydroxyl groups of the codon–anticodon helix, which are expected to be disrupted with a near-cognate codon–anticodon helix. However, a recent structural study showed that G530, A1492 and A1493 form hydrogen bonds in a manner identical with that of both cognate and near-cognate codon–anticodon helices. To understand how the ribosome discriminates between cognate and near-cognate tRNAs, we made 2′-deoxynucleotide and 2′-fluoro substituted mRNAs, which disrupt the hydrogen bonds between the A site codon and G530, A1492 and A1493. Our results show that multiple 2′-deoxynucleotide substitutions in the mRNA substantially inhibit tRNA selection, whereas multiple 2′-fluoro substitutions in the mRNA have only modest effects on tRNA selection. Furthermore, the miscoding antibiotics paromomycin and streptomycin rescue the defects in tRNA selection with the multiple 2′-deoxynucleotide substituted mRNA. These results suggest that steric complementarity in the decoding center is more important than the hydrogen bonds between the A site codon and G530, A1492 and A1493 for tRNA selection.     

Distortion of tRNA upon near-cognate codon recognition on the ribosome

The accurate decoding of the genetic information by the ribosome relies on the communication between the decoding center of the ribosome, where the tRNA anticodon interacts with the codon, and the GTPase center of EF-Tu, where GTP hydrolysis takes place. In the A/T state of decoding, the tRNA undergoes a large conformational change that results in a more open, distorted tRNA structure. Here we use a real-time transient fluorescence quenching approach to monitor the timing and the extent of the tRNA distortion upon reading cognate or near-cognate codons. The tRNA is distorted upon codon recognition and remains in that conformation until the tRNA is released from EF-Tu, although the extent of distortion gradually changes upon transition from the pre- to the post-hydrolysis steps of decoding. The timing and extent of the rearrangement is similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis on the cognate codon. Thus, although the tRNA plays an active role in signal transmission between the decoding and GTPase centers, other regulators of signaling must be involved.     

Decoding the genome: a modified view

Transfer RNA’s role in decoding the genome is critical to the accuracy and efficiency of protein synthesis. Though modified nucleosides were identified in RNA 50 years ago, only recently has their importance to tRNA’s ability to decode cognate and wobble codons become apparent. RNA modifications are ubiquitous. To date, some 100 different posttranslational modifications have been identified. Modifications of tRNA are the most extensively investigated; however, many other RNAs have modified nucleosides. The modifications that occur at the first, or wobble position, of tRNA’s anticodon and those 3′-adjacent to the anticodon are of particular interest. The tRNAs most affected by individual and combinations of modifications respond to codons in mixed codon boxes where distinction of the third codon base is important for discriminating between the correct cognate or wobble codons and the incorrect near-cognate codons (e.g. AAA/G for lysine versus AAU/C asparagine). In contrast, other modifications expand wobble codon recognition, such as U·U base pairing, for tRNAs that respond to multiple codons of a 4-fold degenerate codon box (e.g. GUU/A/C/G for valine). Whether restricting codon recognition, expanding wobble, enabling translocation, or maintaining the messenger RNA, reading frame modifications appear to reduce anticodon loop dynamics to that accepted by the ribosome. Therefore, we suggest that anticodon stem and loop domain nucleoside modifications allow a limited number of tRNAs to accurately and efficiently decode the 61 amino acid codons by selectively restricting some anticodon–codon interactions and expanding others.     

Ribosome hijacking: a role for small protein B during trans‐translation

Tight recognition of codon–anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the ‘tmRNA–SmpB’ system (transfer messenger RNA–small protein B). Remarkably, entry and accommodation of aminoacylated‐tmRNA into stalled ribosomes occur without a codon–anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon–anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for ΔsmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.     

Binding of misacylated tRNAs to the ribosomal A site

To test whether the ribosome displays specificity for the esterified amino acid and the tRNA body of an aminoacyl-tRNA (aa-tRNA), the stabilities of 4 correctly acylated and 12 misacylated tRNAs in the ribosomal A site were determined. By introducing the GAC (valine) anticodon into each tRNA, a constant anticodon.codon interaction was maintained, thus removing concern that different anticodon.codon strengths might affect the binding of the different aa-tRNAs to the A site. Surprisingly, all 16 aa-tRNAs displayed similar dissociation rate constants from the A site. These results suggest that either the ribosome is not specific for different amino acids and tRNA bodies when intact aa-tRNAs are used or the specificity for the amino acid side chain and tRNA body is masked by a conformational change upon aa-tRNA release.     

Alignment/misalignment hypothesis for tRNA selection by the ribosome

Transfer RNAs (tRNAs) are the adaptor molecules that allow the ribosome to decode genetic information during protein synthesis. During decoding, the ribosome must chose the tRNA whose anticodon corresponds to the codon inscribed in the messenger RNA to incorporate the correct amino acid into the growing polypeptide chain. Fidelity is improved dramatically by a GTP hydrolysis event. Information about the correctness of the anticodon must be sent from the decoding center to the elongation factor, EF-Tu, where the GTP hydrolysis takes place. A second discrimination event entails the accommodation of the aminoacyl-tRNA into its fully bound A/A state inside the ribosome. Here, we present a hypothesis for a specific mechanism of signal transduction through the tRNA, which operates during GTPase activation and accommodation. We propose that the rigidity of the tRNA plays an important role in the transmission of the decoding signal. While the tRNA must flex during binding and accommodation, its anisotropic stiffness enables precise positioning of the acceptor arm in the A/T state, the A/A state and the accommodation corridor. Correct alignment will result in optimal GTPase activation and accommodation rates. Incorrect tRNAs, however, whose anticodons are misaligned, will also have acceptor arms that are misaligned, resulting in sub-optimal GTPase activation and accommodation rates. In the case of GTPase activation, it is possible that the misalignment of the acceptor arm affects the rate directly, by altering the conformational change of the switch region of EF-Tu, or indirectly, by changing the alignment of EF-Tu with respect to the sarcin-ricin loop (SRL) of the large ribosomal subunit.     

Testing constraints on rRNA bases that make nonsequence-specific contacts with the codon-anticodon complex in the ribosomal A site

During protein synthesis, interactions between the decoding center of the ribosome and the codon-anticodon complexes maintain translation accuracy. Correct aminoacyl-tRNAs induce the ribosome to shift into a "closed" conformation that both blocks tRNA dissociation and accelerates the process of tRNA acceptance. As part of the ribosomal recognition of cognate tRNAs, the rRNA nucleotides G530 and A1492 form a hydrogen-bonded pair that interacts with the middle position of the codon.anticodon complex and recognizes correct Watson-Crick base pairs. Exchanging these two nucleotides (A530 and G1492) would not disrupt these interactions, suggesting that such a double mutant ribosome might properly recognize tRNAs and support viability. We find, however, that exchange mutants retain little ribosomal activity. We suggest that even though the exchanged nucleotides might function properly during tRNA recruitment, they might disrupt one or more other functions of the nucleotides during other stages of protein synthesis.     

C to U editing of the anticodon of imported mitochondrial tRNA(Trp) allows decoding of the UGA stop codon in Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNA(Trp)(CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNA(Trp) undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.     

Favored and less favored codon–anticodon duplexes arising from the GC codon family box encoding for alanine: some computational perspectives

Alanine is encoded by the four codons of the GC box (GCA, GCG, GCU, and GCC). Known alanine anticodons include the UGC, IGC, and VGC triplets (I = inosine; V = uridine-5-oxyacetic acid). The energy-minimized structures of all possible codon–anticodon combinations involving all the alanine codons GCA, GCG, GCU, and GCC with the alanine anticodons UGC, IGC, and VGC are studied using the AMBER software. Fifteen H-bonded duplex structures arising out of these combinations are studied here, all having Watson–Crick-type base pairs at the first and second codon positions, and a variety of base pairing possibilities at the third (or wobble) position. Structural and stability considerations suggest that some codon–anticodon duplexes would be more favored than others for accommodation during the translation process. The UGC anticodon is predicted to favor the GCA codon for reading, while the GCC codon is least favored. The IGC anticodon would prefer to read the GCC codon, the GCG codon being least favored, while a syn conformer for A in the GCA codon could allow for it to be read. For the VGC anticodon, the GCA codon is predicted to be read most favorably, and the GCC codon least favorably, while a syn conformer for V in the anticodon would allow for the codon GCU to be read through a wobble pair which involves the exocyclic 5-oxyacetate group of V in H-bonding.     

Yeast mitochondrial tRNAIle and tRNAMetm: nucleotide sequence and codon recognition patterns.

The nucleotide sequence of yeast mitochondrial isoleucine- and methionine-elongator tRNA have been determined. Interestingly, long stretches of almost identical nucleotide sequences are found within these two tRNAs and also within the yeast mt tRNAMetf, suggesting that the 3 tRNAs may have arisen from a common ancestor. Both mt tRNAMetm and tRNAIle contain all the structural characteristics which are present in the standard cloverleaf, except that the mt tRNAMetm contains an extra unpaired nucleotide within the base-paired T psi C stem. This rather unusual feature may have an influence on the decoding properties of the C-A-U anticodon of mt tRNAMetm by conferring the ability to translate not only the codon A-U-G but also A-U-A.