Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

The code within the codons

For the first time it is shown that each of the three codon bases has a general correlation with a different, predictable amino acid property, depending on position within the codon. In addition to the previously recognized link between the mid-base and the hydrophobic-hydrophilic spectrum, we show that, with the exception of G, the first base is generally invariant within a synthetic pathway. G--coded amino acids show a different order, being found only at the head of the synthetic pathways. The redundancy of the nature of the third base has a previously unrecognised relationship with molecular weight. The bases U and A (transversions) are associated with the most sharply defined or opposite states in both the first and second position, C somewhat less so or intermediate, anf G neutral. The apparently systematic nature of these relationships has profound implications for the origin of the genetic code. It appears to be the remains of the first language of the cell, predating the tRNA/ribosome system, persisting with remarkably little change at a deeper level of organisation than the codon language.     

Insertion of an extra codon for threonine is a cause of dihydropteridine reductase deficiency.

The mutation in a patient with dihydropteridine reductase deficiency has been located and characterized. Polymerase chain reaction (PCR) was used to amplify the coding sequence of human dihydropteridine reductase from the messenger RNA of skin fibroblasts. Chemical cleavage of mismatches indicated a mismatched thymine and cytosine at approximately 117 and 147 bases, respectively, from the end of the probe. Cloning and sequencing of the mutant PCR products revealed the insertion of the triplet ACT (threonine), after alanine 122 (base 390). Amplification of a small region around this mutation by using genomic DNA as the PCR target indicates that the mutation is completely within an exon. Unequal crossing-over at the second base in the preceding alanine codon and duplication of the bases CTA may be the mechanism of mutagenesis. The cleavage site 147 bases from the end of the probe corresponded to the conversion of guanine to adenine at base 420 (CTG to CTA) and does not alter the code for leucine. This change, which was also seen in another dihydropteridine reductase-deficient child and in a control subject probably represents a common neutral polymorphism.     

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.     

Leftward ribosome frameshifting at a hungry codon.

Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames. Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply. Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame.     

An empirical codon model for protein sequence evolution

In the past, 2 kinds of Markov models have been considered to describe protein sequence evolution. Codon-level models have been mechanistic with a small number of parameters designed to take into account features, such as transition-transversion bias, codon frequency bias, and synonymous-nonsynonymous amino acid substitution bias. Amino acid models have been empirical, attempting to summarize the replacement patterns observed in large quantities of data and not explicitly considering the distinct factors that shape protein evolution. We have estimated the first empirical codon model (ECM). Previous codon models assume that protein evolution proceeds only by successive single nucleotide substitutions, but our results indicate that model accuracy is significantly improved by incorporating instantaneous doublet and triplet changes. We also find that the affiliations between codons, the amino acid each encodes and the physicochemical properties of the amino acids are main factors driving the process of codon evolution. Neither multiple nucleotide changes nor the strong influence of the genetic code nor amino acids' physicochemical properties form a part of standard mechanistic models and their views of how codon evolution proceeds. We have implemented the ECM for likelihood-based phylogenetic analysis, and an assessment of its ability to describe protein evolution shows that it consistently outperforms comparable mechanistic codon models. We point out the biological interpretation of our ECM and possible consequences for studies of selection.     

tRNA slippage at the tmRNA resume codon

The bacterial ribosome does not initiate translation on the mRNA portion of tmRNA; instead translation that had begun on a separate mRNA molecule resumes at a particular triplet on tmRNA (the resume codon). For at least two tRNAs that could pair with both the resume and -2 triplets on mutant tmRNAs, UAA (stop) as the second codon induced high-frequency -2 slippage on the resume codon in the P site. The frameshift product was not detected when the -2 base was altered. Deficiency for ribosomal L9 protein, which affects other cases of frameshifting, had no significant effect. A special feature of this frameshifting is its dependence on a particular context, that of the tmRNA resume codon; it failed on the same sequence in a regular mRNA, and, more strikingly, at the second tmRNA codon. This focuses attention on the peculiar features expected of the slippage-prone state, such as unusual E-site filling, that might make the P-site resume codon:anticodon interaction especially unstable. Keywords: tmRNA; ribosome; frameshift; E site; translation     

Occurrence of nucleotide triplets in genes and secondary structure of the coded proteins

This paper deals with general regularities of nucleotide triplet occurrence in genes of various organisms and their effects on secondary structure of the coded proteins. The strongest general regularity translated into proteins is a predominance of guanine in the first codon position and its deficit in the second codon position. This bias is mostly compensated for by a deficit of thymine in the first and excess of adenine in the second codon positions. These general regularities increase the average amounts of beta sheets and mainly alpha helices in the coded proteins, but they are far from optimal if their only purpose or origin is a promotion of spatial organization of the coded proteins.     

CpG hotspot causes second mutation in codon 408 of the phenylalanine hydroxylase gene

Summary A new mutation has been identified in exon 12 of the gene encoding phenylalanine hydroxylase at codon 408. The single base change from guanine to adenine changes the amino acid arginine to glutamine; thus, the mutation is defined as R408Q. This codon is the site of a mutation known to causes phenylketonuria. Both these mutations are located at the same CpG site.     

A role for initiation codon context in chloroplast translation

To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the -1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii. In prokaryotes, the -1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA. We found that in vivo, none of the chloroplast mutations affected mRNA stability. However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo. Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo. Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.     

Prediction of gene expression under drought stress in spring wheat using codon usage pattern

Spring wheat (Triticum aestivum) is a staple food providing sources of essential proteins for human. In fact, gene expressions of wheat play an important role in growth and productivity that are affected by drought stress. The objective of this work focused on analysis gene feature on spring wheat represented by nucleotide and gene expressions under drought stress. It was found that the higher codon adaptation index was in both wheat root and L-galactono-1, 4-lactone dehydrogenase. It was also found that guanine and cytosine content were high (55.56%) in wheat root. Whereas, guanine and cytosine content were low (41.28%) in L-galactono-1, 4-lactone dehydrogenase. Moreover, the higher relative synonymous codon usage value was observed in codon CAA (1.20), GAA (1.33), GAT (1.00), and ATG (1.00) in wheat root and thus about 62.95% of the total variation in relative synonymous codon was explained by principal component analysis. Additionally, high averages frequency number of codon were (above 15.76) in Met, Lys, Ala, Gly, Phe, Asp, Glu, His, and Tyr; whereas, low averages were in remaining amino acids and majority (90%) of modified relative codon bias values was between 0.40 and 0.90. Shortly, calculations and analysis of codon usage pattern under drought stress would help for genetic engineering, molecular evolution, and gene prediction in wheat studies for developing varieties that associate with drought tolerance.     

Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911.

The proteins expressed by insertion sequence IS911, a member of the widespread IS3 family of elements, have been analyzed. The results indicate that three major species are produced from two consecutive reading frames. A protein of Mr 11,500, ORFA, is synthesized from an upstream reading frame. A larger protein, ORFAB, uses the same initiation codon and is produced by a -1 programmed translational frameshift between orfA and a downstream frame, orfB, whose amino acid sequence shows significant homology with retroviral integrase proteins. The orfB frame is also expressed independently in two alternative forms: the first uses a rare AUU initiation codon in the orfB phase whereas the second appears to initiate in the orfA phase and is produced by a -1 frameshift mechanism similar to that used in ORFAB expression. A specific IS911 integration reaction using a minimal active junction composed of 51 base-pairs of the right inverted repeat and a flanking phase lambda sequence resembling a second end in inverted orientation has been developed to analyze the functions of these proteins by transcomplementation in vivo. The orfA and orfB frames are shown to be essential and production of ORFAB is shown to stimulate integration in this system, suggesting that this fusion protein is the IS911 transposase.     

Sense codon in Escherichia coli are translated in context

The nucleotide frequencies 5' and 3' to the sense codons in highly and weakly expressed genes have been investigated by the chi-squares method. A comparison between the experimental and computer-generated random nucleotide sequences (in which each codon is substituted by a random synonymous one) was made. It was shown that the choice of a particular codon among the synonymous ones in a given position of the gene depends on the three nucleotides 3' and 5' adjacent to the codon in highly expressed genes (the triplet 3' and a single nucleotide 5' to the codons in weakly expressed genes). Concrete patterns for the preferable choice of synonymous codons depending on their contexts are presented. It is suggested that these constraints are related to the efficiency of messenger translation. The constraints on the amino acid sequences of encoded proteins also lead to statistically significant bases in nucleotide frequencies around the sense codons. The biological role of these constraints is discussed.     

Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro.

Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.     

Deletion errors generated during replication of CAG repeats.

Triplet repeat sequence instability is associated with hereditary neurological diseases and with certain types of cancer. Here we study one form of this instability, deletion of triplet repeats during replication of template (CAG)(n)sequences by DNA polymerases. To monitor loss of triplet codons, we inserted (CAG)(9)and (CAG)(17)repeats into the lacZ sequence in M13mp2 and changed one repeat to a TAG codon to yield DNA substrates with colorless plaque phenotypes. Templates containing these inserts within gaps were copied and errors were scored as blue plaque Lac revertants whose DNA was sequenced to determine if loss of the TAG codon resulted from substitutions or deletions. DNA synthesis by either DNA polymerase beta or exonuclease-deficient T7 DNA polymerase produced deletions involving loss of from 1 to 8 of 9 or 15 of 17 repeats. Thus, these polymerases utilize misaligned template-primers containing from 3 to 45 extra template strand nucleotides. Deletion frequencies were much higher than substitution frequencies at the TAG codon in certain repeats, indicating that triplet repeats are at high risk for mutation in the absence of error correction. Proofreading-proficient T7 DNA polymerase generated deletions at 2- to 10-fold lower frequencies than did its exonuclease-deficient derivative. This suggests that misaligned triplet repeat sequences are subject to proofreading, but at reduced efficiency compared to editing of single-base mismatches.     

A method for measuring the non-random bias of a codon usage table

We describe a new statistical method for measuring bias in the codon usage table of a gene. The test is based on the multinomial and Poisson distributions. The method is used to scan DNA sequences and measure the strength of codon preference. For E. Coli we show that the strength of codon preference is related to levels of gene expression. The method can also be used to compare base triplet frequencies with those expected from the base composition. This second type of codon bias test is useful for distinguishing coding from non-coding regions.     

Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli

The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation.