Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem-loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASL(CCCG) and ASL(ACCC) (5'-3' nomenclature) form unpredicted anticodon-codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASL(ACGA) bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon-codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.     

A multivariate study of the relationship between the genetic code and the physical-chemical properties of amino acids

Summary The 20 naturally occurring amino acids are characterized by 20 variables: pK_{NH 2}, pK_COOH, pI, molecular weight, substituent van der Waals volume, seven¹H and¹³C nuclear magnetic resonance shift variables, and eight hydrophobicity-hydrophilicity scales. The 20-dimensional data set is reduced to a few new dimensions by principal components analysis. The three first principal components reveal relationships between the properties of the amino acids and the genetic code. Thus the amino acids coded for by adenosine (A), uracil (U), or cytosine (C) in their second codon position (corresponding to U, A, or G in the second anticodon position) are grouped in these components. No grouping was detected for the amino acids coded for by guanine (G) in the second codon position (corresponding to C in the second anticodon position). The results show that a relationship exists between the physical-chemical properties of the amino acids and which of the A (U), U (A), or C (G) nucleotide is used in the second codon (anticodon) position. The amino acids coded for by G (C) in the second codon (anticodon) position do not participate in this relationship.     

Primordial reading of genetic information

From the consideration of general features of the anticodon loop and stem in tRNA and the properties of present-day translation, we put forward a plausible scenario to explain the evolution of the genetic code from a highly ambiguous triplet code to the present refined decoding system. Our model based on the reading of the code suggests that the anticodon of primordial tRNA could adopt either the 3' or the 5' stacked conformation permitting the formation of the "best two out of three" base pairs, either the first and second codon position or the second and third. Progressive acquisition of precise structural constraint and the modification of bases in the anticodon loop would give way eventually to the less ambiguous "two out of three" reading mechanism having only the 3' stacked conformation. Further adjustments of base composition and modification leads inevitably to the present generalized code. In this way the primordial code encoding 4-8 amino acids or related derivates evolves smoothly to the present code having 20 amino acids.     

Specific interaction between tRNA and its cognate amino acid as detected by circular dichroism and fluorescence spectroscopy

Specific interaction was detected between tRNA and its cognate L-amino acid by circular dichroism (CD) and fluorescence spectroscopy; fluorescence spectral change with saturation was observed when tRNAAsp was titrated only with L-aspartic acid, but not with D-aspartic acid, L- and D-glutamic acids. It was also the case for tRNA2Glu and L-glutamic acid as detected by CD. These results are consistent with the hypothesis that the anticodon, or the complex of four nucleotides (C4N) of the anticodon three bases and the discriminator base in a tRNA (Shimizu, M., J. Mol. Evol. (1982) 18, 297-303) participates in recognition of amino acids.     

tRNA hopping: enhancement by an expanded anticodon.

At a low level wild-type tRNA(1Val) inserts a single amino acid (valine) for the five nucleotide sequence GUGUA which has overlapping valine codons. Mutants of tRNA(1Val) with an insertion of A or U between positions 34 and 35 of their anticodons have enhanced reading of the quintuplet sequences. We propose that this decoding occurs by a hopping mechanism rather than by quintuplet pairing. Such hopping involves disengagement of the paired codon and anticodon with the mRNA slipping two (or more) bases along the ribosomal--peptidyl tRNA complex and subsequently re-pairing at a second codon--the landing site. The mutant with the anticodon sequence 3'CAAU5' 'hops' over the stop codon in the mRNA sequence GUG UAA GUU with the insertion of a single amino acid (valine). In contrast, in reading the same sequence, the mutant with the anticodon 3'CAUU5' hops onto the stop with the insertion of two valine residues. It is likely that in some instances of hopping alternate anticodon bases are used for the initial pairing and at the landing site.     

Complexes of polyadenylic acid and the methyl esters of amino acids

This report includes studies of the binding of the methyl esters of a series of amino acids to polyadenylic acid. The principal data were obtained using proton NMR; however, some additional data were obtained through the study of insoluble complexes and through ultraviolet spectroscopy. The binding constants are in the order Phe>Ile?Leu>Val>Gly, and show a direct correlation with the hydrophobicities of the amino acids. In most cases they are essentially double the binding constants found by Reuben and Polk (1980) for monomeric AMP. All of these amino acids, except Gly, have A as the middle letter of their anticodons, and Phe is the only one with XAA as its only anticodon. It has the anticodon richest in A and has the highest binding constant for A. These results, coupled with other data, continue to support a model of the origin of the code which is based on weak, but selective affinities between amino acids and their anticodons.     

An integrated,structure- and energy-based view of the genetic code

The principles of mRNA decoding are conserved among all extant life forms. We present an integrative view of all the interaction networks between mRNA, tRNA and rRNA: the intrinsic stability of codon–anticodon duplex, the conformation of the anticodon hairpin, the presence of modified nucleotides, the occurrence of non-Watson–Crick pairs in the codon–anticodon helix and the interactions with bases of rRNA at the A-site decoding site. We derive a more information-rich, alternative representation of the genetic code, that is circular with an unsymmetrical distribution of codons leading to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. All tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism, and each anticodon of the sense codons. The multiplicity and complexity of nucleotide modifications at positions 34 and 37 of the anticodon loop segregate meaningfully, and correlate well with the necessity to stabilize AU-rich codon–anticodon pairs and to avoid miscoding in split codon boxes. The evolution and expansion of the genetic code is viewed as being originally based on GC content with progressive introduction of A/U together with tRNA modifications. The representation we present should help the engineering of the genetic code to include non-natural amino acids.     

The complementarity of the chemical parameters of amino acids and anticodons in the genetic code

Chemical language of the genetic code is suggested in which elementary information code units are presented by functional groups of amino acids and nucleotides. Using this language, the existence of correspondence and conformity of chemical parameters of amino acids and of central nucleotides of their anticodons was demonstrated. These findings confirm the idea that the genetic code is determined by chemical properties of amino acids and nucleotides and that this determination is the result of direct specific interactions between amino acids and nucleotide triplets at the stage of the origin of the code. The data obtained reveal primary role of anticodon triplets in the origin of the code. Key role of the central nucleotide in triplets for amino acid coding is confirmed.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

A tRNA aminoacylation system for non-natural amino acids based on a programmable ribozyme

The ability to recognize tRNA identities is essential to the function of the genetic coding system. In translation aminoacyl-tRNA synthetases (ARSs) recognize the identities of tRNAs and charge them with their cognate amino acids. We show that an in vitro evolved ribozyme can also discriminate between specific tRNAs, and can transfer amino acids to the 3' ends of cognate tRNAs. The ribozyme interacts with both the CCA-3' terminus and the anticodon loop of tRNA(fMet), and its tRNA specificity is controlled by these interactions. This feature allows us to program the selectivity of the ribozyme toward specific tRNAs, and therefore to tailor effective aminoacyl-transfer catalysts. This method potentially provides a means of generating aminoacyl tRNAs that are charged with non-natural amino acids, which could be incorporated into proteins through cell-free translation.     

Identifying the ligated amino acid of archaeal tRNAs based on positions outside the anticodon

Proper recognition of tRNAs by their aminoacyl-tRNA synthetase is essential for translation accuracy. Following evidence that the enzymes can recognize the correct tRNA even when anticodon information is masked, we search for additional nucleotide positions within the tRNA molecule that potentially contain information for amino acid identification. Analyzing 3936 sequences of tRNA genes from 86 archaeal species, we show that the tRNAs’ cognate amino acids can be identified by the information embedded in the tRNAs’ nucleotide positions without relying on the anticodon information. We present a small set of six to 10 informative positions along the tRNA, which allow for amino acid identification accuracy of 90.6% to 97.4%, respectively. We inspected tRNAs for each of the 20 amino acid types for such informative positions and found that tRNA genes for some amino acids are distinguishable from others by as few as one or two positions. The informative nucleotide positions are in agreement with nucleotide positions that were experimentally shown to affect the loaded amino acid identity. Interestingly, the knowledge gained from the tRNA genes of one archaeal phylum does not extrapolate well to another phylum. Furthermore, each species has a unique ensemble of nucleotides in the informative tRNA positions, and the similarity between the sets of positions of two distinct species reflects their evolutionary distance. Hence, we term this set of informative positions a “tRNA cipher.” It is tempting to suggest that the diverging code identified here might also serve the aminoacyl tRNA synthetase in the task of tRNA recognition.     

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.     

Conformational relationships between amino acids and their anticodons in the primitive decoding system

Detailed calculations of the conformational characteristics of a primitive decoding system are presented. A penta-nucleotide serves as the primitive tRNA (PIT) with a triplet of primitive anticodon (PAC) in a helical conformation. This molecular moiety has a cleft in the middle. An amino acid can comfortably nestle into the cleft. The conformation of this molecular association is stabilised by a few hydrogen bonds. The stereochemistry of the moiety restricts the conformational possibilities and the sidechain of the amino acid gets oriented at a proper position and in the correct direction to interact intimately with the PAC in the middle of the PIT. The model favours L-amino acids for beta-D-ribonucleotides. The location of the sidechain of the amino acid in the PIT gives a raison d'être for the important features of the organisation of nucleotide triplets for amino acids in the Genetic Code. The interaction of a few key amino acids with the different combinations of bases as PAC sequences has been studied and the stereochemical basis for the selection of the anticodons for amino acids is elucidated.     

The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site.

E. coli threonyl-tRNA synthetase (ThrRS) is a class II enzyme that represses the translation of its own mRNA. We report the crystal structure at 2.9 A resolution of the complex between tRNA(Thr) and ThrRS, whose structural features reveal novel strategies for providing specificity in tRNA selection. These include an amino-terminal domain containing a novel protein fold that makes minor groove contacts with the tRNA acceptor stem. The enzyme induces a large deformation of the anticodon loop, resulting in an interaction between two adjacent anticodon bases, which accounts for their prominent role in tRNA identity and translational regulation. A zinc ion found in the active site is implicated in amino acid recognition/discrimination.     

Genetic code correlations: Amino acids and their anticodon nucleotides

Summary The data here show direct correlations between both the hydrophobicity and the hydrophilicity of the homocodonic amino acids and their anticodon nucleotides. While the differences between properties of uracil and cytosine derivatives are small, further data show that uracil has an affinity for charged species. Although these data suggest that molecular relationships between amino acids and anticodons were responsible for the origin of the code, it is not clear what the mechanism of the origin might have been.     

A biophysical basis for the emergence of the genetic code in protocells

The origin of the genetic code is an abiding mystery in biology. Hints of a ‘code within the codons’ suggest biophysical interactions, but these patterns have resisted interpretation. Here, we present a new framework, grounded in the autotrophic growth of protocells from CO₂ and H₂. Recent work suggests that the universal core of metabolism recapitulates a thermodynamically favoured protometabolism right up to nucleotide synthesis. Considering the genetic code in relation to an extended protometabolism allows us to predict most codon assignments. We show that the first letter of the codon corresponds to the distance from CO₂ fixation, with amino acids encoded by the purines (G followed by A) being closest to CO₂ fixation. These associations suggest a purine-rich early metabolism with a restricted pool of amino acids. The second position of the anticodon corresponds to the hydrophobicity of the amino acid encoded. We combine multiple measures of hydrophobicity to show that this correlation holds strongly for early amino acids but is weaker for later species. Finally, we demonstrate that redundancy at the third position is not randomly distributed around the code: non-redundant amino acids can be assigned based on size, specifically length. We attribute this to additional stereochemical interactions at the anticodon. These rules imply an iterative expansion of the genetic code over time with codon assignments depending on both distance from CO₂ and biophysical interactions between nucleotide sequences and amino acids. In this way the earliest RNA polymers could produce non-random peptide sequences with selectable functions in autotrophic protocells.     

Specificity in the genetic code. The role of nucleotide base-amino acid interaction

The mechanism of the unique and specific association of a given amino acid to its t-RNA is investigated by theoretical methods. Several possible schemes are proposed to explain specificity. The physical forces which act within these mechanisms are illustrated by the computer simulation of probable interactions between glycine and nucleotide bases and base pairs. It is demonstrated that glycine has direct and selective affinities for the nucleotide bases and that these interactions are principally determined by the polar groups. Energies have been calculated for the interaction of glycine with several base pairs. From these, the possibility that specificity arises through direct complexing of an amino acid with its anticodon is evaluated.     

Recognition of tRNAs by aminoacyl-tRNA synthetases: Escherichia coli tRNAMet and E. coli methionyl-tRNA synthetase

In previous work we identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (MetRS) (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. We have now investigated the aminoacylation activity of a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro by using T4 RNA ligase (EC 6.5.1.3). Base substitutions in the wobble position have been found to reduce aminoacylation rates by at least five orders of magnitude. Derivatives having base substitutions in the other two positions of the anticodon are aminoacylated 55-18,500 times slower than normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, which indicates that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of MetRS with functional groups on the nucleotide bases of the anticodon sequence. Several other aminoacyl-tRNA synthetases are known to require one or more anticodon bases for efficient aminoacylation of their tRNA substrates, and data from other laboratories suggest that anticodon sequences may be important for accurate discrimination between cognate and noncoagnate tRNAs by these enzymes.     

The origin of the genetic code: amino acids as cofactors in an RNA world.

The genetic code, understood as the specific assignment of amino acids to nucleotide triplets, might have preceded the existence of translation. Amino acids became utilized as cofactors by ribozymes in a metabolically complex RNA world. Specific charging ribozymes linked amino acids to corresponding RNA handles, which could basepair with different ribozymes, via an anticodon hairpin, and so deliver the cofactor to the ribozyme. Growing of the 'handle' into a presumptive tRNA was possible while function was retained and modified throughout. A stereochemical relation between some amino acids and cognate anticodons/codons is likely to have been important in the earliest assignments. Recent experimental findings, including selection for ribozymes catalyzing peptide-bond formation and those utilizing an amino acid cofactor, hold promise that scenarios of this major transition can be tested.