Nucleic Acids and Protein Biosynthesis

Abstract

Much evidence has accumulated in support of the following model (a working hypothesis) for protein synthesis:Individual amino acids are activated in the presence of ATP, magnesium ions, and specific enzymes [aminoacyl soluble RNA (sRNA) synthetases). to form enzyme-bound aminoacyl adenylates and inorganic pyrophosphate. The carboxyl group of the amino acid is linked to the phosphate group of the adenylate moiety by an anhydride bond. The aminoacyl group of the en zyme-bound complex is transferred to amino acid-specific acceptor sRNA's where it is attached by an ester linkage to the ribose portion of the terminal adenosine residue. The amino acid-binding end of sRNA consists of a cytidyl ate-cytidylate-adenylate (CCA) sequence. Aminoacyl sRNA's react with template-bound ribosomes (polyribosomes, ergosomes) , at specific positions determined by the nucleotide sequence of the template (or messenger) RNA. The aminoacyl sRNA's are specifically aligned in relation to the growing peptide chain, the C-terminal end of which is linked to sRNA. Nucleophilic attack by the a-amino group of the incoming aminoacyl sRNA on the car boxyl carbon atom of peptidyl sRNA results in the formation of a new pep tide bond and release of the sRN A that was previously attached to the end of the growing chain. The polypeptide chain, now containing an additional amino acid residue, isHnked through the new amino acid to its corresponding sRNA. Polyribosome-bound aminoacyl sRNA and peptidyl sRNA are there fore intermediates in protein synthesis. The incorporation of the aminoacyl moiety of aminoacyl sRNA into the growing ribosome-bound peptide chain requires GTP, glutathione, and two enzymes. The messenger and the ribo some then move one coding unit over in relation to each other. A new nucleo tide sequence in messenger RNA is thus placed in position to base-pair properly with the next aminoacyl sRNA. As this process is repeated. the polypeptide chain grows from its N-terminal to its C-terminal residue by the sequential addition of aminoacyl sRNA's. The sequence of amino acids is determined by the nucleotide sequence of messenger RNA; the latter is determined by the deoxynucleotide sequence in DNA, which is transcribed in the form of a complementary polyribonucleotide chain, the messenger.

The Effect of Polyamines on the Aminoacylation of Transfer Ribonucleic Acid

“…The t-RNAs used in these studies were purified with combinations of the following procedures : (a) countercurrent distribution,3 (b) reversed phase column chromatography,' and chromatography on (c) benzoylated DEAEcellulose and (d) DEAE-Sephadex.G The partially purified aminoacyl-t-RNA synthetases were prepared according to procedures described ear lie^.^ The amino acid-acceptor activities of the four purified t-RNAs are given in TABLE 1. It can be seen that each of the t-RNAs except the tyrosine species is more than 80% pure.…”

Toxic Amino Acids: Their Action as Antimetabolites

“…Evidence has been presented that the genetic code is degenerate and also that soluble (transfer)-RNA capable of accepting a specific amino acid is often heterogeneous, being capable of separation into various fractions by column chromatography, countercurrent distribution, and other procedures. The fractions obtained are specific for the various codons established for a particular amino acid (243,244). These different species of sRNA, each specific for the same normal protein amino acid, might discriminate differently between the normal amino acid and its analogs.…”

Protein Synthesis During Fungal Spore Germination II. Aminoacyl-soluble Ribonucleic Acid Synthetase Activities During Germination of Botryodiplodia theobromae Spores

“…phenylalanine could be due to an enzyme deficiency or to low specific activity at one of several steps in the protein synthesis process. It is well established that the first step in the synthesis of protein involves an activation of an individual amino acid by its corresponding aminoacylsoluble ribonucleic acid (sRNA) synthetase enzyme and the subsequent transfer of the activated amino acid to its specific sRNA (10).…”

“…Assay for aminoacyl sRNA formation. The standard assay contained (in a final volume of 0.75 ml) 100 ,pmoles of tris(hydroxymethyl)aminomethane (Tris; pH 7.8, 10 Asmoles of magnesium chloride, 2.5 ,umoles of potassium fluoride, 10 ,moles of adenosine triphosphate (ATP), 0.5 mg of yeast sRNA, 0.3 ,c of "IC-amino acid, and 30 to 200 jAg of enzyme protein. The concentration of added enzyme was such that a doubling of the enzyme concentration produced a doubling of the aminoacylation reaction.…”