Why Were [GADV]-amino Acids and GNC Codons Selected and How Was GNC Primeval Genetic Code Established?

Correspondence relations between codons and amino acids are determined by genetic code. Therefore, genetic code holds a key of the life system composed of genes and protein. According to the GNC-SNS primitive genetic code hypothesis, which I have proposed, it is assumed that the genetic code originated from GNC code. In this article, first, it is discussed from a standpoint of primeval protein synthesis, why four [GADV]-amino acids were selected and used in the first GNC code. Next, it is explained from another standpoint of the most primitive anticodon-stem loop tRNAs (AntiC-SL tRNAs), how four GNCs were selected for the first codons. Furthermore, in the last section of this article, I will explain my idea of how the correspondence relations between four [GADV]-amino acids and four GNC codons were established. Namely, the origin and evolution of the genetic code was discussed comprehensively from several aspects of [GADV]-proteins, [GADV]-amino acids, GNC codons, and anticodon stem-loop tRNAs (AntiC-SL tRNAs), which relate each other to the origin of the genetic code, as integrating GNC code frozen-accident theory, coevolution theory, and adaptive theory on the origin of the genetic code.

How Did Life Emerge in Chemically Complex Messy Environments?

One of the problems that make it difficult to solve the mystery of the origin of life is determining how life emerged in chemically complex messy environments on primitive Earth. In this article, the "chemically complex messy environments" that are focused on are a mixed state of various organic compounds produced via prebiotic means and accumulated on primitive earth. The five factors described below are thought to have contributed to opening the way for the emergence of life: (1) A characteristic inherent in [GADV]-amino acids, which are easily produced via prebiotic means. [GADV] stands for four amino acids, Gly [G], Ala [A], Asp [D] and Val [V], which are indicated by a one-letter symbol. (2) The protein 0th-order structure or a [GADV]-amino acid composition generating water-soluble globular protein with some flexibility, which can be produced even by the random joining of [GADV]-amino acids. (3) The formation of versatile [GADV]-microspheres, which can grow, divide and proliferate even without a genetic system, was the emergence of proto-life. (4) The [GADV]-microspheres with a higher proliferation ability than others were able to be selected. Proto-Darwin evolution made it possible to proceed forward to the creation of a core life system composed of the (GNC)n gene, anticodon stem-loop tRNA or AntiC-SL tRNA (GNC genetic code), and [GADV]-protein. (5) Eventually, the first genuine life with a core life system emerged. Thus, the formation processes of [GADV]-protein and the (GNC)n gene in chemically complex messy environments were the steps to the emergence of genuine life.

The Origin of tRNA Deduced from Pseudomonas aeruginosa 5' Anticodon-Stem Sequence : Anticodon-stem loop hypothesis.

The riddle of the origin of life is unsolved as yet. One of the best ways to solve the riddle would be to find a vestige of the first life from databases of DNA and/or protein of modern organisms. It would be, especially, important to know the origin of tRNA, because it mediates between genetic information and the amino acid sequence of a protein. Here I attempt to find a vestige of the origin and evolution of tRNA from base sequences of Pseudomonas aeruginosa tRNA gene. It was first perceived that 5' anticodon (AntiC) stem sequences of P. aeruginosa tRNA for translation of G-start codon (GNN) are intimately and mutually related. Then, mutual relations among all of the forty-two 5' AntiC stem sequences of P. aeruginosa tRNA were examined. These relationships imply that P. aeruginosa tRNA originated from four anticodon stem-loops (AntiC-SL) translating GNC codons to the corresponding four amino acids, Gly, Ala, Asp and Val (where N is G, C, A, or T). In contrast to the case of AntiC-stem sequence, a mutual relation map could not be drawn with D-, T- and acceptor-stem sequences of P. aeruginosa tRNA. Thus I conclude that the four AntiC-SLs were the first primeval tRNAs.

Evolutionary Steps in the Emergence of Life Deduced from the Bottom-Up Approach and GADV Hypothesis (Top-Down Approach).

It is no doubt quite difficult to solve the riddle of the origin of life. So, firstly, I would like to point out the kinds of obstacles there are in solving this riddle and how we should tackle these difficult problems, reviewing the studies that have been conducted so far. After that, I will propose that the consecutive evolutionary steps in a timeline can be rationally deduced by using a common event as a juncture, which is obtained by two counter-directional approaches: one is the bottom-up approach through which many researchers have studied the origin of life, and the other is the top-down approach, through which I established the [GADV]-protein world hypothesis or GADV hypothesis on the origin of life starting from a study on the formation of entirely new genes in extant microorganisms. Last, I will describe the probable evolutionary process from the formation of Earth to the emergence of life, which was deduced by using a common event-the establishment of the first genetic code encoding [GADV]-amino acids-as a juncture for the results obtained from the two approaches.

Protein ordered sequences are formed by random joining of amino acids in protein 0(th)-order structure, followed by evolutionary process.

Only random processes should occur on the primitive Earth. In contrast, many ordered sequences are synthesized according to genetic information on the present Earth. In this communication, I have proposed an idea that protein 0(th)-order structures or specific amino acid compositions would mediate the transfer from random process to formation of ordered sequences, after formation of double-stranded genes.

[GADV]-protein world hypothesis on the origin of life.

RNA world hypothesis is widely accepted still now, as an idea by which the origin of life might be explained. But, there are many weak points in the hypothesis. In contrast, I have proposed a more reasonable [GADV]-protein world hypothesis or GADV hypothesis, suggesting that life originated from the protein world, which was formed by pseudo-replication of [GADV]-proteins. In this communication, I will discuss about the origin of life from the point of view of the GADV hypothesis.

Pseudo-replication of [GADV]-proteins and origin of life.

The RNA world hypothesis on the origin of life is generally considered as the key to solve the "chicken and egg dilemma" concerning the evolution of genes and proteins as observed in the modern organisms. This hypothesis, however, contains several serious weak points. We have a counterproposal called [GADV]-protein world hypothesis, abbreviated as GADV hypothesis, in which we have suggested that life originated from a [GADV]-protein world, which comprised proteins composed of four amino acids: Gly [G], Ala [A], Asp [D], and Val [V]. A new concept "pseudo-replication" is crucial for the description of the emergence of life. The new hypothesis not only plausibly explains how life originated from the initial chaotic protein world, but also how genes, genetic code, and proteins co-evolved.

Ciliates use both variant and universal genetic codes: evidence of omnipotent eRF1s in the class Litostomatea.

Stop codon reassignments have occurred very frequently in ciliates. In some ciliate species, the universal stop codons UAA and UAG are translated into glutamine, while in some other species, the universal stop codon UGA appears to be translated into cysteine or tryptophan. The class Litostomatea has been hypothesized to be the only group of ciliates using the universal genetic code. However, the hypothesis was based on a statistical analysis of quite small sequence dataset which was insufficient to elucidate the codon usage of the class among such highly deviated phylum. In this study, together with the updated database sequence analysis for the class, we approached the problem of stop codon usage by examining the capacity of the translation termination factor eRF1 for recognizing stop codons. Using in vivo assay systems in budding yeast, we estimated the activity of eRF1 from two litostome species Didinium nasutum and Dileptus margaritifer. The results clearly showed that Didinium and Dileptus eRF1s efficiently recognize all three stop codons. This is the first experimental evidence that strongly supports the hypothesis that litostome ciliates use universal genetic code.

Origin and evolutionary process of the genetic code.

The genetic code plots the relationship between a triplet base sequence on RNA and an amino acid that corresponds to a protein associated with a required function in organisms. Accurate knowledge about the genetic code, including its origin and evolutionary process, would be helpful for determining the causes of genetic disorders and discovering new medical treatments, as well as for understanding the origin of life. This review begins with discussion of several well-known theories on the origin of the genetic code. Then, a GNC-SNS primitive genetic code hypothesis, which we originally proposed, is explained in relation to the weak points of other theories. S and N denote G or C and any of the four bases, respectively. We also introduce our hypothesis of the GADV-protein world hypothesis on the origin of life, where GADV stands for the four amino acids, Gly[G], Ala[A], Asp[D] and Val[V]. Next, we discuss the reason why genetic disorders, which should be triggered by base replacements, are repressed at a low level under the universal genetic code. Finally, we explain the current difficulties we faced in treating genetic disorders, suggesting a prospect for a new type of treatments of these disorders.

Catalytic activities of [GADV]-peptides. Formation and establishment of [GADV]-protein world for the emergence of life.

We have previously postulated a novel hypothesis for the origin of life, assuming that life on the earth originated from "[GADV]-protein world", not from the "RNA world" (see Ikehara's review, 2002). The [GADV]-protein world is constituted from peptides and proteins with random sequences of four amino acids (glycine [G], alanine [A], aspartic acid [D] and valine [V]), which accumulated by pseudo-replication of the [GADV]-proteins. To obtain evidence for the hypothesis, we produced [GADV]-peptides by repeated heat-drying of the amino acids for 30 cycles ([GADV]-P(30)) and examined whether the peptides have some catalytic activities or not. From the results, it was found that the [GADV]-P(30) can hydrolyze several kinds of chemical bonds in molecules, such as umbelliferyl-beta-D-galactoside, glycine-p-nitroanilide and bovine serum albumin. This suggests that [GADV]-P(30) could play an important role in the accumulation of [GADV]-proteins through pseudo-replication, leading to the emergence of life. We further show that [GADV]-octapaptides with random sequences, but containing no cyclic compounds as diketepiperazines, have catalytic activity, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. The catalytic activity of the octapeptides was much higher than the [GADV]-P(30) produced through repeated heat-drying treatments. These results also support the [GADV]-protein-world hypothesis of the origin of life (see Ikehara's review, 2002). Possible steps for the emergence of life on the primitive earth are presented.

Possible steps to the emergence of life: the [GADV]-protein world hypothesis.

Based on the fact that RNA has not only a genetic function but also a catalytic function, the RNA world theory on the origin of life was first proposed about 20 years ago. The theory assumes that RNA was amplified by self-replication to increase RNA diversity on the primitive earth. Since then, the theory has been widely accepted as the most likely explanation for the emergence of life. In contrast, we reached another hypothesis, the [GADV]-protein world hypothesis, which is based on pseudo-replication of [GADV]-proteins. We reached this hypothesis during studies on the origins of genes and the genetic code, where [G], [A], [D], and [V] refer to Gly, Ala, Asp, and Val, respectively. In this review, possible steps to the emergence of life are discussed from the standpoint of the [GADV]-protein world hypothesis, comparing it in parallel with the RNA world theory. It is also shown that [GADV]-peptides, which were produced by repeated dry-heating cycles and by solid phase peptide synthesis, have catalytic activities, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. These experimental results support the [GADV]-protein world hypothesis for the origin of life.

The D-glucosaminate dehydratase alpha-subunit from Pseudomonas fluorescens exhibits thioredoxin reductase activity.

The complete amino acid sequence of the D-glucosaminate dehydratase (GADH) alpha-subunit from Pseudomonas fluorescens was determined by PCR using genomic DNA from P. fluorescens as a template. The alpha-subunit comprises 320 amino acids and has a molecular mass of about 33.9 kDa. The primary structure of the alpha-subunit demonstrates a high similarity to the structures of thioredoxin reductase (TrxR) from many prokaryotes, especially Pseudomonas aeruginosa (identity 85%, positive 91%), Vibrio cholerae (identity 73%, positive 85%), and Escherichia coli (identity 71%, positive 83%). The purified glucosaminate dehydratase alpha(2)-enzyme exhibited NADPH-dependent TrxR activity, while TrxR from E. coli showed pyridoxal 5'-phosphate (PLP)-dependent GADH activity. The TrxR from E. coli suggests that there are three cofactor binding sites, FAD, NADPH, and PLP in the enzyme and that TrxR catalyzes the FAD- and NADPH-dependent oxidation-reduction reaction and the PLP-dependent alpha,beta-elimination reaction.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthetic activities on Escherichia coli SpoT domains.

Escherichia coli SpoT protein, with 702 amino acid residues, is a bifunctional enzyme catalyzing both guanosine 5'-diphosphate 3'-diphosphate (ppGpp) degradation and its synthesis. First, we investigated how many domains are included in SpoT protein, by limited hydrolysis of the protein with serine proteases, alpha-chymotrypsin, and elastase. Based on the results, we deduced that SpoT protein is composed of two major domains, an N-terminal half domain from Met1 to Phe373 and a C-terminal half domain from Glu374 to Asn702 (C-terminal end). In addition, by a further alpha-chymotrypsin digestion, two cleaved sites were found at Arg196 in the N-terminal half domain (D12) and at Lys475 in the C-terminal half domain (D34), to produce four minor domains, D1, D2, D3, and D4. Next, plasmids expressing the two major domains (D12 and D34) and four minor domains (D1, D2, D3, and D4) were constructed. Consequently, the deduced SpoT minor domains as well as the major domains were expressed as stable protein units, except for D4. D4 may also be folded into a stable protein in E. coli cells, since high expression of D4 from a plasmid results in host cell lethality. E. coli relA -, spoT- double null strains expressing D1, D2, and D12 recovered cell growth in M9 minimal medium, but the transformants of D3, D4, and D34 did not grow in the minimal medium. This indicates that ppGpp synthetic activities could be restricted in the N-terminal half domain (D12, D1, and D2).

A novel theory on the origin of the genetic code: a GNC-SNS hypothesis.

We have previously proposed an SNS hypothesis on the origin of the genetic code (Ikehara and Yoshida 1998). The hypothesis predicts that the universal genetic code originated from the SNS code composed of 16 codons and 10 amino acids (S and N mean G or C and either of four bases, respectively). But, it must have been very difficult to create the SNS code at one stroke in the beginning. Therefore, we searched for a simpler code than the SNS code, which could still encode water-soluble globular proteins with appropriate three-dimensional structures at a high probability using four conditions for globular protein formation (hydropathy, alpha-helix, beta-sheet, and beta-turn formations). Four amino acids (Gly [G], Ala [A], Asp [D], and Val [V]) encoded by the GNC code satisfied the four structural conditions well, but other codes in rows and columns in the universal genetic code table do not, except for the GNG code, a slightly modified form of the GNC code. Three three-amino acid systems ([D], Leu and Tyr; [D], Tyr and Met; Glu, Pro and Ile) also satisfied the above four conditions. But, some amino acids in the three systems are far more complex than those encoded by the GNC code. In addition, the amino acids in the three-amino acid systems are scattered in the universal genetic code table. Thus, we concluded that the universal genetic code originated not from a three-amino acid system but from a four-amino acid system, the GNC code encoding [GADV]-proteins, as the most primitive genetic code.

Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens).

A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.

Identification of an indispensable amino acid for ppGpp synthesis of Escherichia coli SpoT protein.

Amino acid substitutions were introduced into a structurally flexible and highly conserved region of Escherichia coli SpoT protein. SpoT protein changed from Asp to Ala at the 293rd position did not restore cell growth of E. coli CF8295 (delta relA, delta spoT) and did not accumulate ppGpp in the cell, suggesting that the Asp293 is indispensable for ppGpp synthesis of the protein.