Editor's note: Citation Classics Commentaries were written by the authors of studies that were some of the most highly cited papers between 1961 and 1975. The essays were originally published between 1977 and 1993 in Current Contents, a publication of the Institute for Scientific Information (ISI), now Thomson Scientific. (ISI was founded by Eugene Garfield, also the founder of The Scientist.) In this essay, published in 1985, Mahlon Hoagland describes his experiments that led to the discovery of transfer RNA.

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The discovery of transfer RNA, described here by Mahlon Hoagland, was critical during a time when an understanding of genetic machinery began to unfold. According to the ISI database, more than 600 papers have cited Hoagland's publication. Hoagland said late last year he considers this finding, along with uncovering the mechanism of amino acid activation in protein synthesis, "my two key discoveries. I can't...

<figcaption>Reference >> M.B. Hoagland, et al., " />
Reference >> M.B. Hoagland, et al., "A soluble ribonucleic acid intermediate in protein synthesis," J Biol Chem, 231:241-57, 1958. Credit: cover image Reprinted from J Biol Chem, 231:241-57, 1958

In 1954 I became one of a group led by Paul Zamecnik at the Massachusetts General Hospital studying protein synthesis. I had just completed a postdoctoral year with Fritz Lipmann in an adjoining lab. Inspired by Lipmann's insights into acyl activation mechanisms and exploiting the Zamecnik group's in vitro rat-liver system, I uncovered the mechanism of amino acid activation in 1955. The enzymatic activity was concentrated in a "soluble" cellular fraction obtained by adjusting cell sap to pH 5 and redissolving the precipitate. In the presence of ATP and amino acids, the fraction vigorously catalyzed the formation of amino acyl adenylates and inorganic pyrophosphate (J Biol Chem, 218:345-58, 1956). Zamecnik's group had already shown that this same fraction was required for incorporation of amino acids into peptide linkages in protein in the presence of microsomes, ATP, and GTP (reviewed in Harvey Lecture, 54:256-81, 1960).

Zamecnik's lab shed the first experimental light on coding while attempting to find if certain of the cell fractions that synthesized protein were also making RNA. To test this, he incubated the pH 5 fraction and microsomes separately with radioactive nucleotides and looked for labeling of acid-precipitable material (RNA and protein). By way of a test for "irrelevant" - or nonspecific - labeling, he carried out parallel incubations with radioactive amino acids. To our surprise, there was both labeling of acid-precipitable material with nucleotides (the - CCA end; Fed Proc, 16:275, 1957) and also considerable labeling by amino acids, particularly in the pH 5 fraction. We found the amino acid label to be in "soluble" mRNA.

Transfer RNA's discovery "offered the first real hope of cracking the code by assigning the nucleotide sequences on tRNA to each of the 20 amino acids." --Mahlon Hoagland

The cited studies indicated that each of the amino acids was bound to the new RNA reversibly and at a separate site. The clincher as to its role in protein synthesis was that after it was charged with amino acids and reisolated, its bound amino acids were rapidly and quantitatively transferred to peptide linkages in protein on ribosomes. And that reaction was dependent on GTP.

Preliminary accounts of these findings appeared in 1957 (Fed Proc, 16:275, 1957; Biochim Biophys Acta, 24:215-6, 1957). Soluble RNA was soon renamed transfer RNA. The work, more fully set forth in the cited 1958 paper, opened the door to an explosion of research characterizing the role of tRNA molecules as amino acid adaptors and as sequencing vehicles via the pairing of their anti-codons with codons of mRNA on ribosomes.

Another reason for the paper's frequent citation was that tRNA's discovery, which came at a time of heated speculation about the nature of the genetic code, offered the first real hope of cracking the code by assigning the nucleotide sequences on tRNA to each of the 20 amino acids. This formidable task was, of course, obviated by the discovery in 1961 that synthetic messenger "RNAs" would promote the synthesis of artificial polypeptides on ribosomes.

My contributions to the understanding of protein synthesis were recognized in 1963 by membership in the American Academy of Arts and Sciences, in 1976 by the award of the Franklin Medal, and in 1984 by membership in the National Academy of Sciences.

Mahlon Hoagland, Office of the President, Worcester Foundation for Experimental Biology, Shrewsbury, Mass. September 9, 1985

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