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Holley, Robert William

HOLLEY, ROBERT WILLIAM

(b. Urbana, Illinois, 28 January 1922; d. Los Gatos, California, 11 February 1993),

organic chemistry, biochemistry, molecular biology, cell biology.

Holley was the first to provide the full sequence of an RNA molecule, alanine transfer RNA, and therefore, indirectly, of a gene. He shared the Nobel Prize in Physiology or Medicine with Marshall Warren Nirenberg and Har Gobind Khorana in 1968 for the characterization of the mechanisms by which the genetic code controls the synthesis of proteins.

Early Life and Training . Robert Holley was one of the four sons of Charles and Viola Holley, both educators. He graduated from Urbana High School in 1938, studied chemistry at the University of Illinois, and received his BA degree in 1942. He studied for his PhD in organic chemistry at Cornell University with Professor Alfred T. Blomquist from 1942 to 1947. His graduate work was interrupted for two years during the war (1944–1946) when he participated with Professor Vincent du Vigneaud at Cornell University Medical College in the first chemical synthesis of penicillin. He married Ann Dworkin in 1945, and they had one son, Frederick.

After two years spent as an American Chemical Society Postdoctoral Fellow at Washington State University, he returned as assistant professor of organic chemistry to the Geneva Experiment Station of Cornell University. He was associate professor there from 1950 to 1957.

The work done during this period differed greatly from the subsequent work that would make Holley famous: characterization of the metabolic transformations of 2,4-dichlorophenoxyacetic acid in bean plants, and identification of the plant hormones, auxins, present in cabbage. From his work on penicillin, Holley established a correlation between the chemical reactivity of amides and their spatial structures, and from these observations he proposed in an article published in Science a general mechanism of action for enzymes that hydrolyze amides, such as proteases, a mechanism which is no longer tenable. This model was inspired by the model of enzymatic catalysis proposed some years before by Linus Pauling—the stabilization of the transition state of the reaction by the enzyme.

These early studies were not without influence on the future activities of Holley, even if they did not point to one particular line of research. They already demonstrate the attention paid by Holley to the contributions that organic chemists can make to biochemistry. Most of all, they familiarized him with the purification procedures that would be of a major importance for his future work—in particular, countercurrent distribution.

Sabbatical Year at Caltech . Holley discovered the world of RNA and protein synthesis during a sabbatical year spent studying with James F. Bonner at the California Institute of Technology. It was increasingly obvious at that time that the characterization of the mechanisms of protein synthesis would come from a full description of the components present in the in vitro protein synthesis systems that had been recently developed in various laboratories, in particular by the group of Paul Zamecnik at the Massachusetts General Hospital. The studies had shown that, before being incorporated into proteins, the amino acids were activated as amino acyl-adenylates by a family of enzymes. More recently, the group of Zamecnik and Mahlon Hoagland had made a puzzling observation: the next step was the attachment of amino acids to RNAs; not the RNAs present in the microsomes and considered for that reason as playing a major role in protein synthesis, but to a new family of small RNAs present in another of the subcellular fractions used for the in vitro synthesis system. Because they were present in a supernatant of ultra-centrifugation, these RNAs were named soluble RNAs. They were shown to be one hundred nucleotides long, but their precise role in protein synthesis remained unknown.

Holley reached the same conclusion by a different approach—the study of the sensitivity to RNAse, the enzyme which degrades RNAs, of the opposite reaction to that of activation of amino acids. At least as far as the amino acid alanine was concerned, soluble RNAs were involved in amino acid activation and incorporation into proteins.

A relation was progressively established between these soluble RNAs and the adapter nucleic acids, which Francis Crick had hypothesized three years before to make the link between the genetic information contained in the nucleic acids and the amino acids constituting the proteins. It pushed the soluble RNAs to the forefront of research. Their characterization increasingly appeared as a sort of Rosetta stone able to reveal the mechanisms of protein synthesis and, for those who were convinced of its existence, to allow the decipherment of the genetic code.

Purification of Alanine tRNA . When Holley returned to Cornell at the Plant, Soil and Nutrition Laboratory of the U.S. Department of Agriculture, he soon undertook the

purification and characterization of tRNA— t for transfer, the new name given to soluble RNA to make its function explicit.

The first step in this seven-year study, which occupied most of Holley’s time, was the purification of this tRNA from yeast. One hundred fifty kilograms of yeast were necessary to yield two hundred grams of mixed tRNAs and one gram of pure alanine tRNA. Two other tRNAs were purified in parallel, tyrosine tRNA and valine tRNA. The technique that proved essential for this purification was countercurrent distribution, a method based on the differential solubility of a molecule between two solvents, which had been designed at the Rockefeller Institute in the 1940s by Lyman C. Craig and David Craig, with whom Holley had worked, and which he had already used to purify auxins.

It was essential to prepare a pure alanine-tRNA fraction in order to proceed to the second part of the work, the sequencing of tRNA. Holley took a gamble on the purity of the fraction that he had obtained in devoting the next four years to this task. Fortunately for him, the gamble paid off.

To determine the sequence of the 77-nucleotide molecule, Holley used the same strategy as the one adopted by Fred Sanger for the protein insulin a few years before: to cut the molecule into different fragments by using enzymes acting at different places, and assemble the different fragments thus obtained, like a puzzle, from the overlapping of the fragments. The work on RNA was made more difficult by the fact that there are only four different nucleotides, compared with twenty amino acids, and this created a lot of ambiguities in the relative positioning of the different fragments. Another unexpected difficulty was the existence of bases with a modified structure and unusual properties, such as the lack of absorption of ultra-violet light, the structure of which had to be characterized. These modifications occur after the synthesis of the tRNA from DNA, at a posttranscriptional stage.

Holley used two different nucleases, pancreatic ribonuclease and takadiastase ribonuclease T1, recently characterized by a Japanese group, which cut at different positions in the sequence, and separated the fragments by chromatography on DEAE-cellulose, a recently designed ion-exchange chromatographic technique which Holley adapted to his purpose. It was not sufficient to determine the full sequence, and a lot of additional tricks had to be used to complete it: progressive degradation of the fragments by an exonuclease, snake venom phosphodiesterase, starting at one extremity of these fragments; a limited and preferential cutting of the tRNA molecule into two fragments obtained by working at low temperature; use of the different characteristics of the two extremities of the tRNA molecule and of the modified bases as markers of unique positions, and so forth.

With the help of “platoons” of graduate students, the full sequence was described in 1965. The position of the anticodon, the site of interaction, by base-pairing, with the messenger RNA, and a model for the secondary structure of the molecule were simultaneously proposed.

A Major Discovery . The value of this experimental achievement was immediately recognized. It was the result of intensive work headed by Holley, from his participation in the discovery of soluble RNA to the final characterization of its structure. The race was intense—a half-dozen structures of tRNA were determined in the next two years, and Holley had won it. The importance of the discovery was rapidly acknowledged: Tracy Sonneborn called it a “marvelous achievement” in Science; Maxine Singer described it as a “formidable job.” Named professor of biochemistry at Cornell University in 1962, Holley became a full professor of biochemistry and molecular biology in 1964 and chairman of the department from 1965 to 1966. He received the Albert Lasker Award in Basic Medical Research in 1965, a first step to the Nobel Prize in 1968, won in conjunction with Marshall Warren Nirenberg and Har Gobind Khorana. He was elected member of the National Academy of Sciences.

The importance of the work was multiple, and the perception of it evolved from its beginnings to its completion. As stated above, tRNAs were first considered as the Rosetta stone leading to the decipherment of the genetic code. But the genetic code was cracked by the use of artificial polynucleotides with a well-determined base composition and sequence from the initial observation made with polyU by Nirenberg. And the anticodon was identified in the tRNA from this previous knowledge. One cause of the degeneracy of the genetic code was explained by the wobble hypothesis proposed by Francis Crick, that is, the imperfect pairing of the third base of the codon with the corresponding one of the anticodon. This hypothesis was proposed from data collected by the use of synthetic polynucleotides, even though it was further supported by observations made on the sequences of tRNAs.

One important result obtained through the sequence of tRNA was the first sequence of a gene. The discovery of Holley was seen as a first step toward knowledge of the full genome. The discourses on what could be expected from this knowledge were not so different from those heard at the end of the twentieth century, when the human genome was sequenced. However, the perception of it as a first step was an illusion: the methodology used for this first study could not provide access to the regulatory sequences that control the expression of genes. In addition, the strategy adopted by Holley could not be easily extended to large RNAs such as messenger RNAs. The technologies for sequencing genes would be invented later, in the mid-1970s, and are of a different nature.

Some of the observations made as Holley’s work progressed—such as the abundance of modified bases— are somehow anecdotal: their physiological meaning is still dubious. The cloverleaf secondary structure proposed by Elizabeth Betty Keller and John Penswick and rapidly adopted by Holley was shown to be true for all tRNAs. It was unduly considered by some researchers as a three-dimensional structure until the characterization of crystallized tRNAs by x-ray diffraction studies done by the group of Aaron Klug showed that this was not the case.

This explains why Holley now occupies both an important and a circumscribed place in the historiography of molecular biology. Retrospectively, while the technological exploit is still worthy of acknowledgment, the consequences of this work for later developments in molecular biology are less important than was initially anticipated. Holley’s discovery was a milestone for his contemporaries, not for his followers.

Control of Cell Division and Cancer . In 1968, there were still many questions pending in the field of protein synthesis: the precise three-dimensional structure of tRNAs and ribosomes was ignored, and the different steps in protein synthesis were still fuzzy. Holley followed the same direction for some years, characterizing the structure of other tRNAs, using indirect chemical techniques to try to elucidate the three-dimensional structure of tRNAs, and turning his efforts to the enzymes involved in the modification of the tRNA bases. But he rapidly abandoned tRNAs, and devoted his efforts to the control of cell division in mammalian cells until the end of his academic career.

In 1966–1967, he spent two years at the recently created Salk Institute for Biological Studies as a National Foundation Postdoctoral Fellow. In 1968 he joined the permanent staff of the Salk Institute as a professor in molecular biology. He was also an adjunct professor at the University of California at San Diego.

Holley was not alone in his movement toward complex biological issues. Many of the founders and heroes of molecular biology made a similar move—Francis Crick, Seymour Benzer, and Gunther Stent toward the study of behavior and the brain, François Jacob and Sydney Brenner toward embryogenesis. Holley preferred to focus on the control of cell division in established mammalian cell lines, probably considering it the best system to characterize and isolate the factors involved in this control.

After some experiments on the extensively studied 3T3 cells, Holley focused his work on an epithelial cell line BSC-1, for the reason that most human cancers are of epithelial origin. He demonstrated that the cells secrete a growth inhibitor, the sequence of which he finally characterized in 1988 after more than ten years of effort.

These twenty years of research on the control of cell division and the characterization of growth inhibitors did not consist simply of data generation. A Nobel Prize winner cannot enter a new field of research without expressing new and general views that strongly oppose previous models. Such was Holley’s attitude. He never accepted the idea that cell division can be limited by “contact inhibition,” signals originating from the physical contact between adjacent cells. For Holley, contact inhibition was only a consequence of a reduced supply of nutrients, due to the fact that their diffusion was limited by the surrounding cells. In a similar way, he was initially reluctant to attribute a major role to growth factors in the control of cell division. In contrast, he emphasized the role of low molecular weight nutrients. In a review article published in 1975 in Nature, he admitted the role of polypeptide hormonelike materials, but once again emphasized the importance of the most common molecules, such as metabolites.

Holley was not interested in the internal, intracellular mechanisms controlling cell division. His only attempt in this direction was the characterization of membrane proteins controlling ion exchange. In 1972 he published in the Proceedings of the National Academy of Sciences a unifying hypothesis concerning the nature of malignant growth. He believed that the crucial alteration leading to malignancy was an alteration in the cell membrane that resulted in an increased internal concentration of nutrients. Over the following years, with the characterization of oncogenes and tumor suppressor genes, the emphasis in cancer research shifted to transformations occurring inside cells and affecting intracellular signaling pathways and gene regulation, and not to membranes as Holley had anticipated. Because of his well-established opinions on the control of cell division and the origin of tumors, Holley was at odds with mainstream research. This prevented him from making a major breakthrough in his new field of research.

BIBLIOGRAPHY

The Salk Institute has decided to create an archival center at the University of California, San Diego.

WORKS BY HOLLEY

With F. P. Boyle, H. K. Durfee, and A. D. Holley. “A Study of the Auxins in Cabbage Using Countercurrent Distribution.” Archives of Biochemistry and Biophysics 32 (1951): 192–199.

“Steric Inhibition of Amide Resonance and Its Possible Significance in Enzyme Action.” Science 117 (1953): 23–25.

“An Alanine-Dependent, Ribonuclease-Inhibited, Conversion of AMP to ATP, and Its Possible Relationship to Protein Synthesis.” Journal of the American Chemical Society 79 (1957): 658–662.

With Jack Goldstein. “An Alanine-Dependent, Ribonuclease-Inhibited Conversion of Adenosine 5'-Phosphate to Adenosine Triphosphate. II. Reconstruction of the System from Purified Components.” Journal of Biological Chemistry 234 (1959): 1765–1768.

With John Robert Penswick. “Specific Cleavage of the Yeast Alanine RNA into Two Large Fragments.” Proceedings of the National Academy of Sciences of the United States of America 53 (1965): 543–546.

With Jean Apgar, George A. Everett, James T. Madison, et al. “Structure of a Ribonucleic Acid.” Science 147 (1965): 1462–1465.

“The Nucleotide Sequence of a Nucleic Acid.” Scientific American 214 (February 1966): 30–39.

“Alanine Transfer RNA.” Nobel Lecture, 12 December 1968. Available from http://nobelprize.org/nobel_prizes/medicine/laureates/1968/holley-lecture.html.

With Josephine A. Kiernan. “‘Contact Inhibition’ of Cell Division in 3T3 Cells.” Proceedings of the National Academy of Sciences of the United States of America 60 (1968): 300–304.

“A Unifying Hypothesis concerning the Nature of Malignant Growth.” Proceedings of the National Academy of Sciences of the United States of America 69 (1972): 2840–2841.

“Control of Growth of Mammalian Cells in Cell Culture.” Nature 258 (1975): 487–490.

With Rosemary Armour, Julia H. Baldwin, et al. “Density-Dependent Regulation of Growth of BSC-1 Cells in Cell Culture: Control of Growth by Serum Factors.” Proceedings of the National Academy of Sciences of the United States of America 74 (1977): 5046–5050.

With Peter Böhlen, Roy Fava, Julia H. Baldwin, et al. “Purification of Kidney Epithelial Cell Growth Inhibitors.” Proceedings of the National Academy of Sciences of the United States of America 77 (1980): 5989–5992.

With Ronald F. Tucker, Gary D. Shipley, and Harold L. Moses. “Growth Inhibitor from BSC-1 Cells Closely Related to Platelet Type Beta Transforming Growth Factor.” Science 226 (1984): 705–707.

With Steven K. Hanks, Rosemary Armour, Julia H. Baldwin, et al. “Amino Acid Sequence of the BSC-1 Cell Growth Inhibitor (Polyergin) Deduced from the Nucleotide Sequence of the cDNA.” Proceedings of the National Academy of Sciences of the United States of Amerca 85 (1988): 79–82.

OTHER SOURCES

Crick, Francis. “Codon-Anticodon Pairing: The Wobble Hypothesis.” Journal of Molecular Biology 19 (1966): 548–555.

Hoagland, Mahlon. Toward the Habit of Truth: A Life in Science. New York: Norton, 1990.

Judson, Horace Freeland. The Eighth Day of Creation: Makers of the Revolution in Biology. Plainview, NY: Cold Spring Harbor Laboratory Press, 1996.

Kay, Lily E. Who Wrote the Book of Life?: A History of the Genetic Code. Stanford, CA: Stanford University Press, 2000. The most complete historical work on the way that led to the “cracking” of the genetic code.

Portugal, Franklin H., and Jack S. Cohen. A Century of DNA: A History of the Discovery of the Structure and Function of the Genetic Substance. Cambridge, MA: MIT Press, 1977.

Rheinberger, Hans-Jörg. Toward a History of Epistemic Things: Synthesizing Proteins in the Test Tube. Stanford, CA: Stanford University Press, 1997.

“Robert W. Holley: The Nobel Prize in Physiology or Medicine 1968.” Available from http://nobelprize.org/nobel_prizes/medicine/laureates/1968/holley-bio.html.

Singer, Maxine F. “1968 Nobel Laureate in Medicine or Physiology.” Science 162 (1968): 433–436.

Sonneborn, Tracy M. “Nucleotide Sequence of a Gene: First Complete Specification.” Science 148 (1965): 1410.

Michel Morange

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Holley, Robert William

Robert William Holley, 1922–93, American biochemist, b. Urbana, Ill., Ph.D. Cornell, 1947. He was a professor at Cornell (1948–68) before he joined (1968) the Salk Institute, and he continued an association with Cornell after 1968. Holley received the 1968 Nobel Prize in physiology or medicine jointly with Har Gobind Khorana and Marshall W. Nirenberg for their interpretation of the genetic code and its function in protein synthesis. Holley is credited with isolating transfer RNA (tRNA) and then determining the sequence and structure of alanine tRNA, which incorporates the amino acid alanine into proteins. Knowledge of the structure of tRNA was key to explaining how proteins are synthesized from messenger RNA.

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