Crick's Wobble Research Articles

A crescendo of competent coding (c3) contains the Standard Genetic Code.

The Standard Genetic Code (SGC) can arise by fusion of partial codes evolved in different individuals, perhaps for differing prior tasks. Such code fragments can be unified into an SGC after later evolution of accurate third-position Crick wobble. Late wobble advent fills in the coding table, leaving only later development of translational initiation and termination to reach the SGC in separated domains of life. This code fusion mechanism is computationally implemented here. Late Crick wobble after C3 fusion (c3-lCw) is tested for its ability to evolve the SGC. Compared with previously studied isolated coding tables, or with increasing numbers of parallel, but nonfusing codes, c3-lCw reaches the SGC sooner, is successful in a smaller population, and presents accurate and complete codes more frequently. Notably, a long crescendo of SGC-like codes is exposed for selection of superior translation. c3-lCw also effectively suppresses varied disordered assignments, thus converging on a unified code. Such merged codes closely approach the SGC, making its selection plausible. For example: Under routine conditions, ≈1 of 22 c3-lCw environments evolves codes with ≥20 assignments and ≤3 differences from the SGC, notably including codes identical to the Standard Genetic Code.

Fitting the standard genetic code into its triplet table

Minimally evolved codes are constructed here; these have randomly chosen standard genetic code (SGC) triplets, completed with completely random triplet assignments. Such "genetic codes" have not evolved, but retain SGC qualities. Retained qualities are basic, part of the underpinning of coding. For example, the sensitivity of coding to arbitrary assignments, which must be < ∼10%, is intrinsic. Such sensitivity comes from the elementary combinatorial properties of coding and constrains any SGC evolution hypothesis. Similarly, assignment of last-evolved functions is difficult because of late kinetic phenomena, likely common across codes. Census of minimally evolved code assignments shows that shape and size of wobble domains controls the code's fit into a coding table, strongly shifting accuracy of codon assignments. Access to the SGC therefore requires a plausible pathway to limited randomness, avoiding difficult completion while fitting a highly ordered, degenerate code into a preset three-dimensional space. Three-dimensional late Crick wobble in a genetic code assembled by lateral transfer between early partial codes satisfies these varied, simultaneous requirements. By allowing parallel evolution of SGC domains, this origin can yield shortened evolution to SGC-level order and allow the code to arise in smaller populations. It effectively yields full codes. Less obviously, it unifies previously studied chemical, biochemical, and wobble order in amino acid assignment, including a stereochemical minority of triplet-amino acid associations. Finally, fusion of intermediates into the final SGC is credible, mirroring broadly accepted later cellular evolution.

Evolution of the Standard Genetic Code

A near-universal Standard Genetic Code (SGC) implies a single origin for present Earth life. To study this unique event, I compute paths to the SGC, comparing different plausible histories. Notably, SGC-like coding emerges from traditional evolutionary mechanisms, and a superior route can be identified. To objectively measure evolution, progress values from 0 (random coding) to 1 (SGC-like) are defined: these measure fractions of random-code-to-SGC distance. Progress types are spacing/distance/delta Polar Requirement, detecting space between identical assignments/mutational distance to the SGC/chemical order, respectively. The coding system is based on selected RNAs performing aminoacyl-RNA synthetase reactions. Acceptor RNAs exhibit SGC-like Crick wobble; alternatively, non-wobbling triplets uniquely encode 20 amino acids/start/stop. Triplets acquire 22 functions by stereochemistry, selection, coevolution, or at random. Assignments also propagate to an assigned triplet’s neighborhood via single mutations, but can also decay. A vast code universe makes futile evolutionary paths plentiful. Thus, SGC evolution is critically sensitive to disorder from random assignments. Evolution also inevitably slows near coding completion. The SGC likely avoided these difficulties, and two suitable paths are compared. In late wobble, a majority of non-wobble assignments are made before wobble is adopted. In continuous wobble, a uniquely advantageous early intermediate yields an ordered SGC. Revised coding evolution (limited randomness, late wobble, concentration on amino acid encoding, chemically conservative coevolution with a chemically ordered elite) produces varied full codes with excellent joint progress values. A population of only 600 independent coding tables includes SGC-like members; a Bayesian path toward more accurate SGC evolution is available.

Crick Wobble and Superwobble in Standard Genetic Code Evolution

Wobble coding is inevitable during evolution of the Standard Genetic Code (SGC). It ultimately splits half of NN U/C/A/G coding boxes with different assignments. Further, it contributes to pervasive SGC order by reinforcing close spacing for identical SGC assignments. But wobble cannot appear too soon, or it will inhibit encoding and more decisively, obstruct evolution of full coding tables. However, these prior results assumed Crick wobble, NN U/C and NN A/G, read by a single adaptor RNA. Superwobble translates NN U/C/A/G codons, using one adaptor RNA with an unmodified 5′ anticodon U (appropriate to earliest coding) in modern mitochondria, plastids, and mycoplasma. Assuming the SGC was selected when evolving codes most resembled it, characteristics of the critical selection events can be calculated. For example, continuous superwobble infrequently evolves SGC-like coding tables. So, continuous superwobble is a very improbable origin hypothesis. In contrast, late-arising superwobble shares late Crick wobble’s frequent resemblance to SGC order. Thus late superwobble is possible, but yields SGC-like assignments less frequently than late Crick wobble. Ancient coding ambiguity, most simply, arose from Crick wobble alone. This is consistent with SGC assignments to NAN codons.

A molecular mechanics study on GA codon box translation

The GA codon box incorporates the two-fold degeneracy of aspartic acid and of glutamic acid. Using the molecular mechanics approach of the AMBER suite, the four codons of the GA box are paired via H-bonding with two aspartic acid anticodons and two glutamic acid anticodons to yield 8 cognate and 11 non-cognate codon-anticodon duplexes. In addition four select non-cognate duplexes between the GA box codons and three alanine anticodons are also studied. These 23 duplexes display a variety of base-pairing possibilities at the wobble position. Cognate duplexes are differentiated from non-cognate duplexes on the grounds of structure and stability (chiefly the former). The results are in line with Crick's wobble hypothesis, and corroborate the observed reading properties of the aspartic acid anticodons GUC and QUC and of the glutamic acid anticodons CUC and SmnUC.

Determinants of translation efficiency and accuracy

Proper functioning of biological cells requires that the process of protein expression be carried out with high efficiency and fidelity. Given an amino-acid sequence of a protein, multiple degrees of freedom still remain that may allow evolution to tune efficiency and fidelity for each gene under various conditions and cell types. Particularly, the redundancy of the genetic code allows the choice between alternative codons for the same amino acid, which, although 'synonymous,' may exert dramatic effects on the process of translation. Here we review modern developments in genomics and systems biology that have revolutionized our understanding of the multiple means by which translation is regulated. We suggest new means to model the process of translation in a richer framework that will incorporate information about gene sequences, the tRNA pool of the organism and the thermodynamic stability of the mRNA transcripts. A practical demonstration of a better understanding of the process would be a more accurate prediction of the proteome, given the transcriptome at a diversity of biological conditions.

Wobble and Superwobble

MOLECULAR BIOLOGY In most cases, more than one triplet codon can specify an amino acid—at one extreme, leucine can be encoded by any of six nucleotide triplets. Degenerate codons tend to vary at the third position, which was the basis for Francis Crick's wobble hypothesis: Each codon must be

Superwobbling facilitates translation with reduced tRNA sets

Some bacterial and most organelle genomes do not encode the full set of 32 tRNA species required to read all codons according to Crick's wobble rules. 'Superwobble', in which a tRNA species with an unmodified U in the wobble position reads all four nucleotides in the third codon position, represents one possible mechanism for how a reduced tRNA set could still suffice. We have tested the superwobble hypothesis by producing knockout mutants for the pair of plastid glycine tRNA genes. Here we show that, whereas the tRNA gene with U in the wobble position is essential, the gene with G in this position is nonessential, demonstrating that the U-containing anticodon can indeed read all four glycine triplets. We also show that the price for superwobbling is a reduced translational efficiency, which explains why most organisms prefer pairs of isoaccepting tRNAs over the superwobbling mechanism.

The wobble hypothesis revisited: Uridine-5-oxyacetic acid is critical for reading of G-ending codons

According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo(5)U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo(5)U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo(5)U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo(5)U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo(5)U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo(5)U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34.

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.

The Molecular Basis of Wobbling: An Alternative Hypothesis

The molecular basis of Crick's wobble hypothesis rests upon the assumption that the wobble base (base closest to the 5′-end of the anticodon) is able to shift its position within the anticodon by ±2·5 Å. Since there are no experimental clues that such shifts do indeed occur, an alternative hypothesis is proposed. It is based upon the observation that a tertiary bond within the tRNA, the C56/G19-bond, is a modified Watson-Crick-bond with the peculiarity of uneven distances between the points of Watson-Crick-contact. Furthermore it is pointed out that C56, as far as its relation to the ribose-phosphate-backbone is concerned, bears a definite resemblance to the wobble base. On these grounds, the C56/G19-bond is discussed as the possible model for all molecular wobble constellations, rendering invalid the need for any shifts within the ribose-phosphate backbone of the anticodonic loop.

Stacking of Crick Wobble pair and Watson-Crick pair: stability rules of G-U pairs at ends of helical stems in tRNAs and the relation to codon-anticodon Wobble interaction.

The occurrence of the noncomplementary G-U base pair at the end of a helix is found to be governed by stacking interactions. As a rule, a G-U pair with G on the 5'-side of a Watson-Crick base pair exhibits strikingly greater stacking overlap with the Watson-Crick base pair than a G-U pair on the 3'-side of a Watson-Crick base pair. The former arrangement is expected to be more stable and indeed is observed 29 times out of 32 in the known transfer RNA molecules. In accordance with this rule, the major wobble base pairs G-U or I-U in codon-anticodon interactions have G or I on the 5'-side of the anticodon. Similarly, in initiator tRNAs, this rule is obeyed where now the G is the first letter of the codon (5'-side). In the situation where U is in the wobble position of the anticodon, it is usually substituted at C(5) andmay also have a 2-thio group and it can read one to four codons depending on its modifications. A G at the wobble position of the anticodon can recognize the two codons ending with U or C and modification of G (unless it is I) does not change its reading properties.

Amino acid substitutions resulting from suppression of nonsense mutations: V. Tryptophan insertion by the Su9+ gene, a suppressor of the UGA nonsense triplet

The Su9 + suppressor gene of Escherichia coli enables the UGA nonsense triplet to code for tryptophan. The implications of this finding in light of the predictions of Crick's wobble hypothesis are discussed.

An examination of the energetics of Crick's wobble hypothesis

In order to account for the observed amino acid coding, Crick postulated the possible existence of certain “wobble configurations” of base-pairs in the third position of the codon-anticodon triplet. Some of these are examined here as follows. The configuration of these pairs is varied and the total interaction energy between the bases calculated by a previously described method. The configuration which corresponds to the minimum energy is selected. All but one configuration are similar to the postulated “wobble configurations” of Crick. The one exception constitutes a redundancy in coding and thus does not affect Crick's postulate. The energies found for these configurations are similar to those previously computed for the usual Watson-Crick pairs, and thus by analogy sufficient to stabilize the “wobble configurations”. An additional result of the computations is to support Nash and Bradley's assertion that the minima of the electrostatic energy and of the total energy occur for very similar configurations.