Genetic-code evolution for protein synthesis with non-natural amino acids

The genetic encoding of synthetic or “non-natural” amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG “stop” triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites.     

A reexamination of the propensities of amino acids towards a particular secondary structure: classification of amino acids based on their chemical structure

The correlation between the primary and secondary structures of proteins was analysed using a large data set from the Protein Data Bank. Clear preferences of amino acids towards certain secondary structures classify amino acids into four groups: α-helix preferrers, strand preferrers, turn and bend preferrers, and His and Cys (the latter two amino acids show no clear preference for any secondary structure). Amino acids in the same group have similar structural characteristics at their Cβ and Cγ atoms that predicts their preference for a particular secondary structure. All α-helix preferrers have neither polar heteroatoms on Cβ and Cγ atoms, nor branching or aromatic group on the Cβ atom. All strand preferrers have aromatic groups or branching groups on the Cβ atom. All turn and bend preferrers have a polar heteroatom on the Cβ or Cγ atoms or do not have a Cβ atom at all. These new rules could be helpful in making predictions about non-natural amino acids.

The genetic code as a periodic table

Summary The contemporary genetic code is reflective of a significant correlation between the properties of amino acids and their anticodons in a periodic manner. Almost all properties of amino acids showed a greater correlation to anticodonic than to codonic dinucleoside monophosphate properties. The polarity and bulkiness of amino acid side chains can be used to predict the anticodon with considerable confidence. The results are most consistent with predictions of the direct interaction and ambiguity reduction hypotheses for the origin of the genetic code.     

Does the Genetic Code Have A Eukaryotic Origin?

In the RNA world, RNA is assumed to be the dominant macromolecule performing most, if not all, core "house-keeping" functions. The ribo-cell hypothesis suggests that the genetic code and the translation machinery may both be born of the RNA world, and the introduction of DNA to ribo-cells may take over the informational role of RNA gradually, such as a mature set of genetic code and mechanism enabling stable inheritance of sequence and its variation. In this context, we modeled the genetic code in two content variables-GC and purine contents-of protein-coding sequences and measured the purine content sensitivities for each codon when the sensitivity (% usage) is plotted as a function of GC content variation. The analysis leads to a new pattern-the symmetric pattern-where the sensitivity of purine content variation shows diagonally symmetry in the codon table more significantly in the two GC content invariable quarters in addition to the two existing patterns where the table is divided into either four GC content sensitivity quarters or two amino acid diversity halves. The most insensitive codon sets are GUN (valine) and CAN (CAR for asparagine and CAY for aspartic acid) and the most biased amino acid is valine (always over-estimated) followed by alanine (always under-estimated). The unique position of valine and its codons suggests its key roles in the final recruitment of the complete codon set of the canonical table. The distinct choice may only be attributable to sequence signatures or signals of splice sites for spliceosomal introns shared by all extant eukaryotes.     

The most probable number of blocks for the partitions of the set of codons could have determined the number of standard amino acids

Given a genetic code formed by 64 codons, we calculate the number of partitions of the set of encoding amino acid codons. When there are 0-3 stop codons, the results indicate that the most probable number of partitions is 19 and/or 20. Then, assuming that in the early evolution the genetic code could have had random variations, we suggest that the most probable number of partitions of the set of encoding amino acid codons determined the actual number 20 of standard amino acids.     

Source of amino acids for tRNA acylation in growing chicks

Summary Specific radioactivity in three amino acid compartments was examined in broiler chicks following a flooding dose of leucine or phenylalanine. In general, specific radioactivity of leucine and phenylalanine in deproteinated plasma (SA_e) and tissue (SA_i) compartments, exceeded that in acylated-tRNA (SA_t). In most tissues, SA_e and SA_i rapidly reached a similar peak level by 5 min followed by a slow decline for the next 30 minutes. Many tissues (eg. GI tract, liver, skin, and thigh) failed to maintain equilibrium between SA_e and SA_i over time. More metabolically active tissues, such as GI and liver had the greatest differences between these compartments. The difference between SA_e and SA_i for both leucine and phenylalanine were due to SA_i decreasing faster than SA_e, indicating dilution with unlabelled amino acids from proteolysis. Plasma and tissue specific radioactivity overestimated tRNA specific radioactivity by as much as 5 and 2.8 fold using leucine or 2.7 and 1.4 fold using phenylalanine, respectively. These data suggest that intracellular compartmentation of protein metabolism and the coupling of protein degradation and synthesis occur, in vivo.     

Determination of mutation trend in proteins by means of translation probability between RNA codes and mutated amino acids

In this study, we estimate the translation probability to amino acid from RNA codon. With the determined 183 translation probabilities and amino-acid composition of eight highly mutated proteins, we construct the theoretical distributions of mutated amino acids in these proteins and then compare them with their actual distributions affected by mutations. Thereafter we trace the pattern of translation probabilities from RNA codons to mutated amino acids of 1053 point missense mutations. Finally, we statistically conclude that the natural mutation trend goes along the theoretical translation probability.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

Summary The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (t_m) for poly(A) poly(U) and poly(I)poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of t_m at low concentrations (1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala>Gly>Ser>Asn>Pro>Met, Val>Ile, Leu for poly(A) poly(U) and Ala, Gly, Ser>Asn>Pro>Val>Ile, Met, Leu for poly(I)poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

The origin of the biologically coded amino acids

Biology uses essentially 20 amino acids for its coded protein enzymes, representing a very small subset of the structurally possible set. Most models of the origin of life suggest organisms developed from environmentally available organic compounds. A variety of amino acids are easily produced under conditions which were believed to have existed on the primitive Earth or in the early solar nebula. The types of amino acids produced depend on the conditions which prevailed at the time of synthesis, which remain controversial. The selection of the biological set is likely due to chemical and early biological evolution acting on the environmentally available compounds based on their chemical properties. Once life arose, selection would have proceeded based on the functional utility of amino acids coupled with their accessibility by primitive metabolism and their compatibility with other biochemical processes. Some possible mechanisms by which the modern set of 20 amino acids was selected starting from prebiotic chemistry are discussed.     

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents

Summary. Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR. Received June 11, 2000 Accepted September 26, 2000     

Codon Usage Bias and Mutation Constraints Reduce the Level of ErrorMinimization of the Genetic Code

Studies on the origin of the genetic code compare measures of the degree of error minimization of the standard code with measures produced by random variant codes but do not take into account codon usage, which was probably highly biased during the origin of the code. Codon usage bias could play an important role in the minimization of the chemical distances between amino acids because the importance of errors depends also on the frequency of the different codons. Here I show that when codon usage is taken into account, the degree of error minimization of the standard code may be dramatically reduced, and shifting to alternative codes often increases the degree of error minimization. This is especially true with a high CG content, which was probably the case during the origin of the code. I also show that the frequency of codes that perform better than the standard code, in terms of relative efficiency, is much higher in the neighborhood of the standard code itself, even when not considering codon usage bias; therefore alternative codes that differ only slightly from the standard code are more likely to evolve than some previous analyses suggested. My conclusions are that the standard genetic code is far from being an optimum with respect to error minimization and must have arisen for reasons other than error minimization.[Reviewing Editor: Martin Kreitman]     

Use of Chou-Fasman amino acid conformational parameters to analyze the organization of the genetic code and to construct protein genealogies

Summary Chou-Fasman parameters, measuring preferences of each amino acid for different conformational regions in proteins, were used to obtain an amino acid difference index of conformational parameter distance (CPD) values. CPD values were found to be significantly lower for amino acid exchanges representing in the genetic code transitions of purines, GA than for exchanges representing either transitions of pyrimidines, CU, or transversions of purines and pyrimidines. Inasmuch as the distribution of CPD values in these non GA exchanges resembles that obtained for amino acid pairs with double or triple base differences in their underlying codons, we conclude that the genetic code was not particularly designed to minimize effects of mutation on protein conformation. That natural selection minimizes these changes, however, was shown by tabulating results obtained by the maximum parsimony method for eight protein genealogies with a total occurrence of 4574 base substitutions. At the beginning position of the codons GA transitions were in very great excess over other base substitutions, and, conversely, CU transitions were deficient. At the middle position of the codons only fast evolving proteins showed an excess of GA transitions, as though selection mainly preserved conformation in these proteins while weeding out mutations affecting chemical properties of functional sites in slow evolving proteins. In both fast and slow evolving proteins the net direction of transitions and transversions was found to be from G beginning codons to non-G beginning codons resulting in more commonly occurring amino acids, especially alanine with its generalized conformational properties, being replaced at suitable sites by amino acids with more specialized conformational and chemical properties. Historical circumstances pertaining to the origin of the genetic code and the nature of primordial proteins could account for such directional changes leading to increases in the functional density of proteins.In order to further explore the course of protein evolution, a modified parsimony algorithm was developed for constructing protein genealogies on the basis of minimum CPD length. The algorithm's ability to judge with finer discrimination that in protein evolution certain pathways of amino acid substitution should occur more readily than others was considered a potential advantage over strict maximum parsimony. In developing this CPD algorithm, the path of minimum CPD length through intermediate amino acids allowed by the genetic code for each pair of amino acids was determined. It was found that amino acid exchanges representing two base changes have a considerably lower average CPD value per base substitution than the amino acid exchanges representing single base changes. Amino acid exchanges representing three base changes have yet a further marked reduction in CPD per base change. This shows how extreme constraining effects of stabilizing selection can be circumvented, for by way of intermediate amino acids almost any amino acid can ultimately be substituted for another without damage to an evolving protein's conformation during the process.     

Relationship between G + C in silent sites of codons and amino acid composition of human proteins

Summary We have investigated the relationship between the G + C content of silent (synonymous) sites in codons and the amino acid composition of encoded proteins for approximately 1,600 human genes. There are positive correlations between silent site G + C and the proportions of codons for Arg, Pro, Ala, Trp, His, Gln, and Leu and negative ones for Tyr, Phe, Asn, Ile, Lys, Asp, Thr, and Glu. The median proteins coded by groups of genes that differ in silent-site G + C content also differ in amino acid composition, as do some proteins coded by homologous genes. The pattern of compositional change can be largely explained by directional mutation pressure, the genetic code, and differences in the frequencies of accepted amino acid substitutions; the shifts in protein composition are likely to be selectively neutral.Offprint requests to: D.W. Collins     

Reactions of 4 methylphenyl isocyanate with amino acids

Arylisocyanates are important intermediates in the chemical industry. Amongst the main damage after low levels of isocyanate exposure are lung sensitization and asthma. Protein adducts of isocyanates might be involved in the aetiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. To develop methods for the quantitation of protein adducts we reacted 4 methylphenyl isocyanate 4MPI with the tripeptide valyl glycyl glycine and with single amino acids yielding N 4 methylphenyl carbamoyl L valyl glycyl glycine 4MPI Val Gly Gly, N 4 methylphenyl carbamoyl L valine 4MPI Val, N 4 methylphenyl carbamoyl L aspartic acid 4MPI Asp, N acetyl S 4 methylphenyl carbamoyl L cysteine 4MPI AcCys, N acetyl N 4 methylphenyl carbamoyl lysine 4MPI AcLys, N acetyl O 4 methylphenyl carbamoyl tyrosine 4MPI AcTyr and N acetyl O 4 methylphenyl carbamoyl D,L serine 4MPI AcSer. The hydrolysis of the adducts was tested under acidic and basic conditions, to obtain the maximum yield of 4 methylaniline 4MA. The isocyanates were hydrolysed for 1 h, 3h and 24h at 100 C with 6 M HCl in and or 0.1 M NaOH at room temperature, following methods applied for the analyses of biological samples of arylisocyanate exposed workers. In addition, we applied a new protocol: the adducts were hydrolyzed for 1-24 h in 0.3 M NaOH at 100 C. The hydrolysates were analysed using HPLC with UV detection and quantified against the internal standard, 4 fluoroaniline or 4 chloroaniline. 4MA was obtained with the best yields using 0.3M NaOH; after 24 h all amino acid adducts were cleaved under these conditions. Acid hydrolysis of 4MPI Val and 4MPI Asp yielded the respective hydantoins 3 4 methylphenyl 5 isopropyl 1,3 imidazoline 2,4 dione and 2 1 4 methylphenyl 2,5 dioxoperhydro 4 imidazolyl acetic acid. For future studies, we propose to hydrolyse biological samples with 0.3 M NaOH at 100 C to release the maximum amount of 4MA from the adducts. However, in biological samples from workers, hydrolysable adducts can also result from arylamine exposure. Therefore, we propose to analyse the N terminal adducts of isocyanates with blood protein to distinguish between arylamine and arylisocyanate exposure.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Secretory signal sequence non-optimal codons are required for expression and export of beta-lactamase

In this study we altered the codon usage in the signal sequence of the bla gene, encoding β-lactamase in Escherichia coli. Changing all of the thirteen non-optimal codons to optimal lowered expression 4-fold as measured by minimum inhibitory concentration (MIC) to the β-lactam antibiotic ampicillin. The difference in ampicillin resistance was reduced at 28 °C compared to expression at 37 °C, suggesting that the optimised bla allele is misfolded and degraded by heat-shock regulated proteases. A screen was carried out, designed specifically to identify revertants with changes in codon usage resulting in higher MIC to ampicillin. The nine revertants revealed by this method all had optimal to non-optimal codon changes in the signal sequence. These results, and those of our previous study with maltose binding protein model system, confirm that non-optimal codons are important for expression and export of secretory proteins via both the SecB-dependent and -independent pathways.     

The regularities of the changes of amino acid physico-chemical properties within the genetic code

Summary All the codons of the genetic code can be arranged into the closed one-step mutation ring, containing three periods of the same sequence of mutations (2,3,3,3,1,3,3,3,1,3,3,3,1,3,3,3,2,3,3,3). The codons of Gly play a role of the connecting element between the end of the third, and the beginning of the first period of the genetic code. The reactivity of amino acids, expressed by the reaction rates of aminolysis reaction of N-hydroxysuccinimide esters of protected amino acids with p-anisidine, changes periodically with the respect to the mutation periods of the genetic code. Chou-Fasman P as well as P conformational parameters of amino acids, and also the compositional frequencies of amino acids in proteins, demonstrate the pseudosymmetry pattern with respect to the center of one-step mutation ring, which is situated between Thr ACY and ACR codons.     

Synthesis of symmetric and asymmetric diamides of citric acid and amino acids

Summary A convenient method for the synthesis of symmetric and asymmetric diamides of amino acids including DOPA and citric acid from 2-tert-butyl-1,3-di(N-hydroxysuccinimidyl)citrate and 1-tert-butyl-2,3-di(N-hydroxysuccinimidyl)citrate is described.Abbreviations AcOtBu tert-butyl acetate - i-Bu iso-butyl - tBu tert-butyl - Bzl benzyl - p-OH-Bzl p-hydroxybenzyl - m,p-(OH)₂-Bzl m,p-dihydroxybenzyl - DCCI dicyclohexylcarbodiimide - Et ethyl - Me methyl - Su succinimidyl - SuOH N-hydroxysuccinimide - Ph phenyl     

Determination of amino acid pairs sensitive to variants in human copper-transporting ATPase 2

In this study, we use our probabilistic approach to analyze the amino acid pairs in human copper-transporting ATPase 2 (ATP7B) in order to determine which amino acid pairs are more sensitive to 125 variants from missense mutant human ATP7B. The results show 97.6% of 125 variants occur at randomly unpredictable amino acid pairs, which account for 80.9% of amino acid pairs in ATP7B, and the chance of occurring of variant is about 9 times higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, the randomly unpredictable amino acid pairs are more sensitive to variants in human ATP7B.     

Distribution of mapping points of 20 amino acids in the tetrahedral space

Summary Based on the genetic codes and a simple theorem for the geometrical property of the regular tetrahedron, each amino acid is mapped onto a unique point in a 3-dimensional tetrahedral space. The distribution of the 20 mapping points for 20 amino acids is studied in detail. It is found that the mapping points for the hydrophobic and hydrophilic amino acids are distributed at distinct regions in the 3-dimensional space. A plane separating the two kinds of points satisfactorily based on the Fisher's algorithm has been calculated. It is shown that the codons coding for the hydrophobic amino acids are constituted dominantly by the bases of keto group, i.e., G and T. While the codons coding for the hydrophilic amino acids are constituted dominantly by the bases of amino group, i.e., A and C. The biological implication of the mapping points and the separating plane has been discussed in some details.