Amino-acid Sequence of Thermolysin

The following three articles describe how chemical and X-ray analyses have been combined to elucidate the structure of thermolysin. This first article describes the determination of the amino-acid sequence of this metalloendopeptidase.     

Genetic Code Mutations: The Breaking of a Three Billion Year Invariance

The genetic code has been unchanging for some three billion years in its canonical ensemble of encoded amino acids, as indicated by the universal adoption of this ensemble by all known organisms. Code mutations beginning with the encoding of 4-fluoro-Trp by Bacillus subtilis, initially replacing and eventually displacing Trp from the ensemble, first revealed the intrinsic mutability of the code. This has since been confirmed by a spectrum of other experimental code alterations in both prokaryotes and eukaryotes. To shed light on the experimental conversion of a rigidly invariant code to a mutating code, the present study examined code mutations determining the propagation of Bacillus subtilis on Trp and 4-, 5- and 6-fluoro-tryptophans. The results obtained with the mutants with respect to cross-inhibitions between the different indole amino acids, and the growth effects of individual nutrient withdrawals rendering essential their biosynthetic pathways, suggested that oligogenic barriers comprising sensitive proteins which malfunction with amino acid analogues provide effective mechanisms for preserving the invariance of the code through immemorial time, and mutations of these barriers open up the code to continuous change.     

Amino-acid Sequence of Substance P

IN 1931 von Euler and Gaddum¹, studying the tissue distribution of acetylcholine, found that brain and intestine contained a substance that stimulated contraction of the isolated rabbit jejunum and caused transient hypotension when injected intravenously into anaesthetized rabbits. These effects could not be ascribed to acetylcholine, for they were not prevented by the previous administration of atropine. The initial studies were made on crude acid alcohol extracts of equine brain and intestine, dried in powder form. The active principle in the preparation was later referred to as substance P (P for powder) and this non-committal term subsequently achieved widespread acceptance in the literature, in the absence of any clearly definable biological role for the compound (or compounds) involved.     

Amino-acid Sequence of Human Placental Lactogen

AN earlier report from this laboratory¹ described striking homologies in the amino-acid composition of the peptides obtained from trypsin cleavage of human placental lactogen (HPL) and human growth hormone (HGH) and established a firm biochemical basis for the similarities in biological and immunological activity of the two hormones. The intrachain disulphide bonds were placed in similar locations and the carboxyl-terminal amino-acid sequences were shown to be identical except for one out of fourteen residues². In other studies^3,4, the amino-terminal sequence was determined and noted to be homologous for eleven of the first seventeen residues. We now report the complete amino-acid sequence of HPL and a comparison with the revised structure for HGH recently reported⁴. These two protein hormones are strikingly similar in structure, being identical in 85% of the corresponding positions. Of twenty-eight amino-acid substitutions, twenty-five are favoured changes and all but two of the twenty-five can be explained in terms of a single base change in the triplet codon⁵.     

遗传密码扩充技术

遗传密码的扩充打破了天然生命体基因只能编码天然氨基酸的限制.遗传密码扩充技术使人们可以将具有特殊性质的非天然氨基酸通过基因表达到生物活体的蛋白质中.该文对遗传密码扩充的原理、方法及意义进行简述.     

Phenylalanine-Binding RNAs and Genetic Code Evolution

We isolated RNAs by selection–amplification, selecting for affinity to Phe–Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets. Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70% of the 105 sequenced RNAs. The K _D for the strongest sites is ≈50 μM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites. However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 × 10⁻¹¹. Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg, Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during an era of direct RNA–amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during later code evolution.     

On the Evolution of the Standard Genetic Code: Vestiges of Critical Scale Invariance from the RNA World in Current Prokaryote Genomes

Herein two genetic codes from which the primeval RNA code could have originated the standard genetic code (SGC) are derived. One of them, called extended RNA code type I, consists of all codons of the type RNY (purine-any base-pyrimidine) plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. In order to test if putative nucleotide sequences in the RNA World and in both extended RNA codes, share the same scaling and statistical properties to those encountered in current prokaryotes, we used the genomes of four Eubacteria and three Archaeas. For each prokaryote, we obtained their respective genomes obeying the RNA code or the extended RNA codes types I and II. In each case, we estimated the scaling properties of triplet sequences via a renormalization group approach, and we calculated the frequency distributions of distances for each codon. Remarkably, the scaling properties of the distance series of some codons from the RNA code and most codons from both extended RNA codes turned out to be identical or very close to the scaling properties of codons of the SGC. To test for the robustness of these results, we show, via computer simulation experiments, that random mutations of current genomes, at the rates of 10⁻¹⁰ per site per year during three billions of years, were not enough for destroying the observed patterns. Therefore, we conclude that most current prokaryotes may still contain relics of the primeval RNA World and that both extended RNA codes may well represent two plausible evolutionary paths between the RNA code and the current SGC.     

Genetic Code Variations in Mitochondria: tRNA as a Major Determinant of Genetic Code Plasticity

Characteristic features of tRNA such as the anticodon sequence and modified nucleotides in the anticodon loop are thought to be crucial effectors for promoting or restricting codon reassignment. Our recent findings on basepairing rules between anticodon and codon in various metazoan mitochondria suggest that the complete loss of a codon is not necessarily essential for codon reassignment to take place. We postulate that a possible competition between two tRNAs with cognate anticodon sequences towards the relevant codon to be varied has a potential role in codon reassignment. Our proposition can be viewed as an expanded version of the codon capture theory proposed by Osawa and Jukes (J Mol Evol 28: 271–278, 1989). Received: 28 December 2000 / Accepted: 12 March 2001     

Coding Rules for Amino Acids in the Genetic Code: The Genetic Code is a Minimal Code of Mutational Deterioration

Coding rules for amino acids in the genetic code are discussed from the point that the genetic code is a minimal code ofmutational deterioration. The global mutational deterioration(GMD) function is defined through several parameters describingsingle base mutations and amino acid distances. The problem ofsearching for the global minimum of the GMD function is discussedin some detail. From GMD minimization under initial constraintswe have succeeded in deducing the standard genetic code.     

Error-reducing Structure of the Genetic Code Indicates Code Origin in Non-thermophile Organisms

During the RNA World, organisms experienced high rates of genetic errors, which implies that there was strong evolutionary pressure to reduce the errors’ phenotypical impact by suitably structuring the still-evolving genetic code. Therefore, the relative rates of the various types of genetic errors should have left characteristic imprints in the structure of the genetic code. Here, we show that, therefore, it is possible to some extent to reconstruct those error rates, as well as the nucleotide frequencies, for the time when the code was fixed. We find evidence indicating that the frequencies of G and C in the genome were not elevated. Since, for thermodynamic reasons, RNA in thermophiles tends to possess elevated G+C content, this result indicates that the fixation of the genetic code occurred in organisms which were either not thermophiles or that the code’s fixation occurred after the rise of DNA. Supplementary Materials Original data and programs are available at the author’s web site: .     

Studying Genetic Code by a Matrix Approach

Following Petoukhov and his collaborators, we use two length n zero-one sequences, α and β, to represent a length n genetic sequence ((a) || (b)){alphachoosebeta} so that the columns of ((a) || (b)){alphachoosebeta} have the following correspondence with the nucleotides: C ~ (0 || 0)Csim{0choose0} , U ~ (1 || 0)Usim{1choose0} , G ~ (1 || 1)Gsim{1choose1} , A ~ (0 || 1)Asim{0choose1} . Using the Gray code ordering to arrange α and β, we build a 2 ⁿ ×2 ⁿ matrix C _n including all the 4 ⁿ length n genetic sequences. Furthermore, we use the Hamming distance of α and β to construct a 2 ⁿ ×2 ⁿ matrix D _n . We explore structures of these matrices, refine the results in earlier papers, and propose new directions for further research.     

The Evolution of the Genetic Code Revisited

The evolution of the genetic code in terms of the adoption of new codons has previously been related to the relative thermostability of codon–anticodon interactions such that the most stable interactions have been hypothesised to represent the most ancient coding capacity. This derivation is critically dependent on the accuracy of the experimentally determined stability parameters. A new set of parameters recently determined for B-DNA reveals that the codon–anticodon pairs for the codes in non-plant mitochondria on the one hand and prokaryotic and eukaryotic organisms on the other can be unequivocally divided into two classes – the most stable base steps define a common code specified by the first two bases in a codon while the less stable base steps correlate with divergent usage and the adoption of a 3-letter code. This pattern suggests that the fixation of codons for A, G, P, V, S, T, D/E, R may have preceded the divergence of the non-plant mitochondrial line from other organisms. Other variations in the code correlate with the least stable codon–anticodon pairs. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Metabolic Basis for the Self-Referential Genetic Code

An investigation of the biosynthesis pathways producing glycine and serine was necessary to clarify an apparent inconsistency between the self-referential model (SRM) for the formation of the genetic code and the model of coevolution of encodings and of amino acid biosynthesis routes. According to the SRM proposal, glycine was the first amino acid encoded, followed by serine. The coevolution model does not state precisely which the first encodings were, only presenting a list of about ten early assignments including the derivation of glycine from serine—this being derived from the glycolysis intermediate glycerate, which reverses the order proposed by the self-referential model. Our search identified the glycine-serine pathway of syntheses based on one-carbon sources, involving activities of the glycine decarboxylase complex and its associated serine hydroxymethyltransferase, which is consistent with the order proposed by the self-referential model and supports its rationale for the origin of the genetic code: protein synthesis was developed inside an early metabolic system, serving the function of a sink of amino acids; the first peptides were glycine-rich and fit for the function of building the early ribonucleoproteins; glycine consumption in proteins drove the fixation of the glycine-serine pathway.     

Optimization Models and the Structure of the Genetic Code

The codon assignment of the quasi-universal genetic code can be assumed to have resulted from the evolutionary pressures that prevailed when the code was still evolving. Here, we review studies of the structure of the genetic code based on optimization models. We also review studies that, from the structure of the code, attempt to derive aspects of the primordial circumstances in which the genetic code froze. Different rationales are summarized, compared with experimental data, discussed in the context of the transition from a RNA world to a DNA-protein world, and linked to the emergence of the last universal common ancestor.