Masataka Kinjo

Enzymatic Synthesis and Some Properties of a Model Primitive tRNA

SummaryA model primitive tRNA with the nucleotide sequence GGCCAAAAAAAGGCCp was synthesized using T4 RNA ligase. The nucleotide sequence of this newly synthesized oligonucleotide was confirmed by ladder analysis of several enzymatic digestion products. The secondary structure of the oligonucleotide was examined by comparison of the products of its digestion by single- and double-strand-specific nucleases with those of the digestion of the intermediate oligonucleotide GGCCAAAAAAAOH. The results indicated that the two GGCC segments of the 5′ and 3′ ends of the model tRNA may form base pairs in solution. The same conclusion was derived from the result of affinitycolumn chromatography of the model oligonucleotide. When32pGGCCAAAAAAAGGCCOH was passed through a poly(U)-agarose column, about 70% of the applied sample bound to the poly(U)-agarose. In contrast, when the model oligonucleotide was passed through a poly(C)-agarose column, only 15% of the sample bound to the poly(C)-agarose. These results indicate that the newly synthesized oligonucleotide adopts a hairpin structure in solution. Two aspects of a potential biological activity of the synthetic model tRNA were examined. It was found that the oligonucleotide can bind to poly(U)-programmed 30S ribosomes and is recognized by Qβ replicase as a template for RNA synthesis.

The selection and coexistence of a plural number of primitive tRNAs and the origin of the genetic code.

Simulation experiments on Darwinian evolution of primitive tRNAs in coacervate droplets were carried out using four kinds of colored marbles in a computer system. Plural numbers of specified primitive tRNAs were selected and coexisted continuously under the suitable conditions. We suggest that these conditions simulate the real process of selection and coexistence of primitive tRNAs and the origin of the genetic code.

Differential coupling efficiency of chemically activated amino acid to tRNA

SummaryInteraction based on possible chemical affinity of an amino acid for tRNA was examined as a model for the aminoacylation of primitive tRNA without aid of an enzyme system. Two types of reaction were carried out and compared. One was the acyl linkage of amino acid to the 5′-terminal phosphate of a tRNA activated as an imidazolide. The other was the incorporation of an amino acid activated as an imidazolide into 2′(3′)-hydroxyl groups of intact tRNA. Both types of reaction indicated that none of the amino acids tested had any selectivity for the tRNAs examined. However, the rates of reaction with a given tRNA were different among amino acids. In the second type of reaction, amino acids were found mainly at loop-out regions of tRNA, but not at either its 5′- or 3′-terminal sites

EFFECT OF POLYNUCLEOTIDES AND A BASIC PROTEIN ON THE CONDENSATION OF PHENYLALANYL ADENYLATE

The effect of polyuridylate and of histone on the oligomerization of phenylalanyl adenylate was tested. Polyuridylate which pairs with adenine under suitable conditions showed no effect. Histone increased the rate of the polymerization whereas a neutral protein, albumin, had no effect. Simultaneous presence of nucleotides diminished the effect of histone. The implication of these results on the prebiotic polypeptide formation is discussed.

Synthesis of hydrocarbons under presumed prebiotic conditions using high-frequency discharge.

SummaryVarious hydrocarbons were synthesized by high-frequency discharge in a primordial terrestrial model atmosphere. The products were extracted by benzene or methanol and analyzed by GC-MS. The mean carbon chain length of the hydrocarbons formed by the discharge through pure CH4 gas was less than 6. Benzene was also obtained. Some isomers were obtained for each of the hydrocarbons containing a given number of carbons. When a small amount of C2H2 was added to the CH4, longer chain compounds were formed, as compared with discharge in CH4 only. However, when the amount of C2H2 was increased, unextractable high molecular weight compounds were produced. The amounts of products decreased as the mixing ratio of CO2 to CH4 increased. No hydrocarbons were detected when the ratio of CO2/CH4 exceeded 1.

Formation of amino acids from models of Titan and more oxidized atmospheres

Protein and non-protein amino acids were synthesized following hydrolysis of products obtained by high frequency discharge techniques applied to model atmospheres consisting of N2 as a nitrogen source together with CH4 and/or CO2 as a carbon source. Highest yields were obtained in the absence of CO2 and from mixtures rich in CH4. Amino acids would indeed be expected on the frozen surface of Titan with its CH4−N2 atmosphere.

Primitive Transfer RNA and Origin of Darwinian System

Stepwise progress of the relation between amino acids and nucleotides in the origin of protein synthesis mechanism is speculated. As an experimental support for the speculation, the effect of polynucleotide and polypeptide on the polypeptide formation is tested. Basic protein, histone, promotesthe reaction of polypeptide formation from aminoacyl adenylate. The relation between origin and development of genetic code and incerasing of invariable site in protein in the course of molecular evolution is discussed. Learning system is thought to have some analogy with Darwinian evolution.

Catalytic mechanism of histone in peptide formation from phenylalanyl adenylate

Catalytic action of histone on peptide formation from phenylalanyl adenylate was studied. A peptide bond formation from phenylalanyl adenylate releases two protons. The proton release was increased in the presence of histone at a higher concentration. The catalytic action seems to be due to basic circumstance induced by histone, since (1) basic condition enhanced the proton release; (2) basic amino acid did not enhance the proton release.