James C. Lacey

Genetic code correlations: Amino acids and their anticodon nucleotides

SummaryThe data here show direct correlations between both the hydrophobicity and the hydrophilicity of the homocodonic amino acids and their anticodon nucleotides. While the differences between properties of uracil and cytosine derivatives are small, further data show that uracil has an affinity for charged species. Although these data suggest that molecular relationships between amino acids and anticodons were responsible for the origin of the code, it is not clear what the mechanism of the origin might have been.

Aminoacyl transfer from an adenylate anhydride to polyribonucleotides

SummaryImidazole catalysis of phenylalanyl transfer from phenylalanine adenylate anhydride to the hydroxyl groups of homopolyribonucleotides was investigated as a chemical model of the biochemical aminoacylation of tRNA. Imidazole catalyzed transfer of phenylalanine to poly (U) increases from pH 6.5 to 7.7 and decreases above pH 7.7. At pH 7.7 approximately 10% of the phenylalanyl residues are transferred to poly (U). At pH 7.1, transfer to poly (U) was five times as great as to poly (A) and transfer to a poly (A) poly (U) double helix was negligible. At pH 7.1 approximately 45 mole percent linkages to poly (U) were monomeric phenylalanine; the remainder of the linkages were peptides of phenylalanine. The number of linkages and their lability to base and neutral hydroxylamine indicates that phenylalanine and its peptides are attached as esters to the 2′ hydroxyl groups throughout poly (U) and the 2′ (3) hydroxyl groups at the terminus of poly (U). These results do model the contemporary process of aminoacyl transfer to tRNA and continue to suggest that a histidine residue is in the active site of aminoacyl-tRNA-synthetases.

Hydrolytic properties of phenylalanyl- and N-acetylphenylalanyl adenylate anhydrides

We have used a novel spectrophotometric method to study the hydrolysis of N-acetylphenylalanyl adenylate anhydride (AcPhe-AMP) and phenylalanyl-adenylate anhydride (Phe-AMP) at low concentrations (10−5 M), 25 °C, constant buffer concentration (0.05 M), and as a function of pH. While Phe-AMP is susceptible principally to attack by OH−, with two different rates depending on whether the α-amino group of the amino acid is protonated or not, the AcPhe-AMP is susceptible to acid decomposition as well. At pHs 4–8, the Phe-AMP hydrolyzes faster than the AcPhe-AMP, but at pH less than 4 or pH greater than 8, the blocked form hydrolyzes faster. Both forms are also attacked by H2O, and at the same rate. Moreover, the hydrolysis of Phe-AMP is shown to be greatly catalyzed by carbonate, although the AcPhe-AMP is not subject to such catalysis. The rate laws for the various mechanisms and the activation energies for the hydrolyses at pH 7.1 are given.

Aminoacyl transfer: Peptide synthesis and other properties of an amino acid imidazolide

Abstract N- (Glycyl)-imidazole was examined as a chemical model of a possible active site intermediate of enzymes involved in peptide bond formation. The preparation, purification and properties of N- (glycyl)-imidazole were investigated. Preparation was from glycinium trifluoroacetate and 1,1′-carbonyl-diimidazole in dimethylsulfoxide. The product contained significant amounts of N- (glycylglycyl)-imidazole and unreacted glycine which were removed by ion-exchange chromatography. The product was extremely stable to hydrolysis below pH 3, and of constant stability from pH 3–8 becoming more unstable above pH 9. In comparison, its derivative N- (acetylglycyl)-imidazole is much less stable at all pH values and is maximally stable at pH 7 and dramatically less stable at both higher and lower pH values. Peptide formation from the N- (glycyl)-imidazole was favored between pH 6 and pH 9 and decreased rapidly above and below this range. The yield and size of peptides was concentration dependent both increasing with concentration.

Chemical esterification of 5'-AMP occurs predominantly at the 2' position.

SummaryWe describe experiments here which show that chemical esterification of 5′-adenylic acid (5′-AMP) withN-acetylD-orL-phenylalanine (Ac-D- or Ac-L-Phe) imidazolide occurs principally, if not exclusively, at the 2′ position. Furthermore, in experiments with the formation of the 2′–3′ diester with butyric acid andN-acetyl glycine (Ac-Gly), we found the second esterification was also predominantly at the 2′ position. This means that mixed diesters can be predictably prepared with the positions of the substituents known. The results are consistent with a model for the preferential catalytic synthesis ofL-based peptides via a 2′–3′ diester intermediate of purine monoribonucleotides.

Ribonucleic acids may be catalysts for the preferential synthesis ofl-amino acid peptides: A minireview

SummaryThis minireview is a summary of the basic concepts and pieces of experimental evidence supporting a hypothesis that suggests a mechanism whereby purine monoribonucleotides havingd-ribose may be able to preferentially catalyze the synthesis ofl-amino acid peptides. The proposed mechanism involves a 2′–3′ diaminoacyl intermediate and the preference accrues from several factors that favor thel-isomer, principally for hydrophobic amino acids. Although the hypothesis has not been fully tested, some crucial evidence has been published. Other pieces of evidence are now being submitted or are in press for publication and still other experiments, principally on the step of peptide bond formation, are in the process of being carried out. The purpose of a review at this point is to present the hypothesis to the scientific community in hopes of generating discussion, suggestions, and evaluation by other workers. Should the hypothesis prove correct, it may represent the most primitive and fundamental relationship between the nucleic acid and protein systems. In addition, it would represent another important example of the catalytic ability of RNA.

Studies of the chemical basis of the origin of protein synthesis Initiation and direction of peptide growth

SummaryThe data presented in this paper show that the ease of non-enzymatic activation of carboxylic acids by ATP at pH 5 varies directly with the pKa of the carboxyl group, and is consistent with the idea that it is the protonated form of the carboxyl group which participates in the activation reaction. Consequently, since most N-blocked amino acids have higher pKas than do their unblocked forms, they are activated more readily, and we have demonstrated that this principle applies to peptides as well,which are activated more rapidly than single amino acids. We propose that this fact may be partly responsible for the origin of two important features still observed in contemporary protein synthesis: (1) initiation in prokaryotes is accomplished with an N-blocked amino acid, and (2) elongation in all living systems occurs at the carboxyl end of the growing peptide.

Complexes of polyadenylic acid and the methyl esters of amino acids

This report includes studies of the binding of the methyl esters of a series of amino acids to polyadenylic acid. The principal data were obtained using proton NMR; however, some additional data were obtained through the study of insoluble complexes and through ultraviolet spectroscopy. The binding constants are in the order Phe>Ile⩾Leu>Val>Gly, and show a direct correlation with the hydrophobicities of the amino acids. In most cases they are essentially double the binding constants found by Reuben and Polk (1980) for monomeric AMP. All of these amino acids, except Gly, have A as the middle letter of their anticodons, and Phe is the only one with XAA as its only anticodon. It has the anticodon richest in A and has the highest binding constant for A. These results, coupled with other data, continue to support a model of the origin of the code which is based on weak, but selective affinities between amino acids and their anticodons.

Comparative rates of esterification of 5′-AMP with hydrophobic amino acids: Relevance to the genetic-code assignments

SummaryWe have continued our program aimed at understanding the origin and evolution of the genetic code and the process of protein synthesis by comparing the rates of esterification of 5′-AMP by a series of hydrophobic N-acetylamino acids. The reaction clearly shows differences in reaction rate (AcPhe>AcLeu>AcVal>AcIle) among the amino acids having A as middle letter of their anticodons. However, there were no significant differences in reaction rate between AcLeu, AcNorleu, and Ac-α-aminobutyric acid, and AcGly reacted faster than all of these and AcPhe. Consequently, this simple reaction with AMP can distinguish only among those amino acids that actually have A as the middle anticodonic nucleotide. The relevance of these studies to the origins of the process of protein synthesis and of the genetic code is discussed in conjunction with results from other studies of a similar nature.We have continued our program aimed at understanding the origin and evolution of the genetic code and the process of protein synthesis by comparing the rates of esterification of 5′-AMP by a series of hydrophobic N-acetylamino acids. The reaction clearly shows differences in reaction rate (AcPhe>AcLeu>AcVal>AcIle) among the amino acids having A as middle letter of their anticodons. However, there were no significant differences in reaction rate between AcLeu, AcNorleu, and Ac-α-aminobutyric acid, and AcGly reacted faster than all of these and AcPhe. Consequently, this simple reaction with AMP can distinguish only among those amino acids that actually have A as the middle anticodonic nucleotide. The relevance of these studies to the origins of the process of protein synthesis and of the genetic code is discussed in conjunction with results from other studies of a similar nature.

Was there a universal tRNA before specialized tRNAs came into existence

It is generally true that evolving systems begin simply and become more complex in the evolutionary process. For those who try to understand the origin of a biochemical system, what is required is the development of an idea as to what simpler system preceeded the present one. Here we present an hypotheis that a universal tRNA molecule, capable of reading many codons may have preceeded the appearance of individual tRNAs. Evidence seems to suggest that this molecule may have been derived from a common ancestor of the contemporary 5S rRNAs and tRNAs.