Wobble base-pairing in codon-anticodon interactions: a theoretical modelling study

The Crick wobble hypothesis attributes the phenomenon of codon degeneracy to a certain impreciseness of pairing between the third base of the codon and the first base of the anticodon. This theoretical study investigates the pairing properties of some wobble bases, including both, observed and unobserved pairs. Some wobble base-pairs are predicted to follow the Watson-Crick pairs in configuration and pairing facility, while others deviate from this norm. The observed U:V pair is unique in that a pairing configuration may be suggested for it wherein the hydrogen-bonding involves the exocyclic 5-carboxymethoxy group of V. By comparing the theoretical data on the configurations of these pairs with the evidence for their existence/non-existence in nature, some guidelines emerge for differentiating between observed and unobserved base pairs on the basis of the pairing configuration.     

Structure of a purine-purine wobble base pair in the decoding center of the ribosome

Here we report the crystal structures of I.C and I.A wobble base pairs in the context of the ribosomal decoding center, clearly showing that the I.A base pair is of an I(anti).A(anti) conformation, as predicted by Crick. Additionally, the structures enable the observation of changes in the anticodon to allow purine-purine base pairing, the 'widest' base pair geometry allowed in the wobble position.     

A conformational rationale for the wobble behaviour of the first base of the anticodon triplet in tRNA

We present a conformational rationale for wobble behaviour of the first base in the anticodon triplet of tRNA and hence for the well-known degeneracy of the genetic code. The U-turn hydrogen bond plays an important role in the structure of the anticodon arm and particularly for the anticodon triplet to be in a geometry suitable for the process of recognition in the adaptor-mediated synthesis of proteins. This hydrogen bond in turn precludes a hydrogen bond between the first two sugars of the anticodon triplet, allowing the first base to wobble, while it facilitates one between the second and third sugars of the triplet, positioning these bases for the standard base-pairing with the codon. This neatly explains why there is a degeneracy in the code and why a RNA happens to be the adaptor for protein synthesis. Relevent conformational calculations are presented in support of the theory.     

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.     

Classification of the possible pairs between the first anticodon and the third codon positions based on a simple model assuming two geometries with which the pairing effectively potentiates the decoding complex

Crick's wobble theory states that some specific pairs between the bases at the first position of the anticodon (position 34) and the third position of the codon (position III) are allowed and the others are disallowed during the correct codon recognition. However, later researches have shown that the pairing rule, or the wobble rule, is different from the supposed one. Despite the continuing efforts including computer-aided model building studies and analyses of three-dimensional structures in the crystals of the ribosomes, the structural backgrounds of the wobble rule are still unclear. Here, I classify the possible pairs into 6 classes according to the increases accompanying the formation of the pairs in the potential productivity of the decoding complex on the basis of a simple model that was originally proposed previously and is refined here. In the model, the conformation with the base at position 34 displaced toward the minor groove side from the position for the Watson-Crick pairs is supposed to be equivalent to the conformation with the Watson-Crick pairs. It is also reasoned and supposed that some weak pairs may sometimes be allowed depending on the structural context. It is demonstrated that most of the experimental results reported so far are consistent with the model. I discuss on which experimental facts can be reasoned with the model and which need further explanations. I expect that the model will be a good basis for further understanding of the wobble rule and its structural backgrounds.     

Stacking geometry for non‐canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion‐corrected DFT‐D study

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson‐Crick base pairs, different orientations of the bases to form hydrogen‐bonded non‐canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non‐canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non‐canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non‐canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll‐slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 328–338, 2015.     

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.     

Identification and codon reading properties of 5-cyanomethyl uridine,a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA₂^Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA₂^Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.     

New information content in RNA base pairing deduced from quantitative analysis of high-resolution structures

Non-canonical base pairs play important roles in organizing the complex three-dimensional folding of RNA. Here, we outline methodology developed both to analyze the spatial patterns of interacting base pairs in known RNA structures and to reconstruct models from the collective experimental information. We focus attention on the structural context and deformability of the seven pairing patterns found in greatest abundance in the helical segments in a set of well-resolved crystal structures, including (i–ii) the canonical A·U and G·C Watson–Crick base pairs, (iii) the G·U wobble pair, (iv) the sheared G·A pair, (v) the A·U Hoogsteen pair, (vi) the U·U wobble pair, and (vii) the G·A Watson–Crick-like pair. The non-canonical pairs stand out from the canonical associations in terms of apparent deformability, spanning a broader range of conformational states as measured by the six rigid-body parameters used to describe the spatial arrangements of the interacting bases, the root-mean-square deviations of the base-pair atoms, and the fluctuations in hydrogen-bonding geometry. The deformabilties, the modes of base-pair deformation, and the preferred sites of occurrence depend on sequence. We also characterize the positioning and overlap of the base pairs with respect to the base pairs that stack immediately above and below them in double-helical fragments. We incorporate the observed positions of the bases, base pairs, and intervening phosphorus atoms in models to predict the effects of the non-canonical interactions on overall helical structure.     

Wobble position modified nucleosides evolved to select transfer RNA codon recognition: a modified-wobble hypothesis.

While recognized that some wobble exists in the base pairing of the first base of the tRNA anticodon with the third of the codon, specific base modifications have evolved to select particular codons. This modified-wobble theory would be exemplified by a single codon recognition imposed on the anticodon by modification of the tRNA wobble position nucleoside.     

RNA structure and dynamics: A base pairing perspective

RNA is now known to possess various structural, regulatory and enzymatic functions for survival of cellular organisms. Functional RNA structures are generally created by three-dimensional organization of small structural motifs, formed by base pairing between self-complementary sequences from different parts of the RNA chain. In addition to the canonical Watson–Crick or wobble base pairs, several non-canonical base pairs are found to be crucial to the structural organization of RNA molecules. They appear within different structural motifs and are found to stabilize the molecule through long-range intra-molecular interactions between basic structural motifs like double helices and loops. These base pairs also impart functional variation to the minor groove of A-form RNA helices, thus forming anchoring site for metabolites and ligands. Non-canonical base pairs are formed by edge-to-edge hydrogen bonding interactions between the bases. A large number of theoretical studies have been done to detect and analyze these non-canonical base pairs within crystal or NMR derived structures of different functional RNA. Theoretical studies of these isolated base pairs using ab initio quantum chemical methods as well as molecular dynamics simulations of larger fragments have also established that many of these non-canonical base pairs are as stable as the canonical Watson–Crick base pairs. This review focuses on the various structural aspects of non-canonical base pairs in the organization of RNA molecules and the possible applications of these base pairs in predicting RNA structures with more accuracy.     

The difference in the type of codon-anticodon base pairing at the ribosomal P-site is one of the determinants of the translational rate

By utilizing an enzymatically reconstructed tRNA variant containing an altered anticodon sequence, we have examined the different biochemical behavior of translation between the Watson-Crick type and the wobble type base pair interactions at the first anticodon position. We have found that the Watson-Crick type base pair has an advantage in translation in contrast to the wobble type base pair by comparing the efficiency of transpeptidation of native tRNA(Phe) (anticodon; GmAA) with its variant tRNA (anticodon; AAA) in the poly(U)-programmed ribosome system. Thomas et al. [Proc. Natl. Acad. Sci. U.S. (1988) 85, 4242-4246] showed that the wobble codon at the ribosomal A-site accepted its cognate tRNA less efficiently than the Watson-Crick base pairing codon. We report here that the wobble interaction at the ribosomal P-site also affected the rate of translation. This variable translational rate may be a mechanism of gene regulation through preferential codon usage.     

Codon reading by tRNAAla with modified uridine in the wobble position

tRNAs reading four-codon families often have a modified uridine, cmo(5)U(34), at the wobble position of the anticodon. Here, we examine the effects on the decoding mechanism of a cmo(5)U modification in tRNA(1B)(Ala), anticodon C(36)G(35)cmo(5)U(34). tRNA(1B)(Ala) reads its cognate codons in a manner that is very similar to that of tRNA(Phe). As Ala codons are GC rich and Phe codons AU rich, this similarity suggests a uniform decoding mechanism that is independent of the GC content of the codon-anticodon duplex or the identity of the tRNA. The presence of cmo(5)U at the wobble position of tRNA(1B)(Ala) permits fairly efficient reading of non-Watson-Crick and nonwobble bases in the third codon position, e.g., the GCC codon. The ribosome accepts the C-cmo(5)U pair as an almost-correct base pair, unlike third-position mismatches, which lead to the incorporation of incorrect amino acids and are efficiently rejected.     

tRNA1Ser(G34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons

The tRNA1Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli. Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon. The tRNA1Ser(G34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons. This means that the formation of G-A or G-G pair allowed at the wobble position and these base pairs are noncanonical. The translational efficiency of the tRNA1Ser(G34) for UCA or UCG codon depends on the 2'-O-methylation of the C32 (Cm). The 2'-O-methylation of C32 may give rise to the space necessary for G-A or G-G base pair formation between the first position of anticodon and the third position of codon.     

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.     

Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria.

Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn)and tRNA(Lys)were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys)corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met)corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system. tRNA(Ser)GCU corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or G at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system.