Leon Heppel and the Early Days of RNA Biochemistry

Today, machines turn out the sequence of a million DNA bases in a day. Fifty years ago, when the chemical and biochemical tools for studying DNA and RNA were at best rudimentary, such a machine was unimaginable. Then, the cutting edge was Erwin Chargaff's demonstration, in 1948, that the base composition of DNA could be reliably determined. His discovery that all DNAs contain equal amounts of adenine and thymine and similarly of guanine and cytosine depended on applying two recent developments: partition chromatography and the absorption spectra of nucleic acid constituents. DNA chemistry took a huge step forward when James D. Watson and Francis Crick, using Chargaff's data, constructed the double helical model of DNA. In contrast, insights into RNA structure lagged behind, thus hampering progress in understanding how DNA carries out its genetic function. At the beginning of the 1950s, studies on RNA structure in the United States came mainly from the laboratories of Waldo Cohn and C. E. Carter. Progress was slow because of the difficulty of determining whether the internucleotide bond was 5′ to 3′ or 5′ to 2′, because the primitive methods available could not cope with the instability of RNA compared with DNA, and because of the still unrecognized existence of several types of RNA. The impetus for further advances in DNA and RNA biochemistry emerged not from structural investigations but from studies of the enzymology of phosphate-containing coenzymes and nucleotides. Much of that activity was spurred by a group of extraordinary biomedical scientists, who partly through the accidents of World War II assignments to the United States Public Health Service found themselves at war's end together at the still fledgling National Institutes of Health (NIH) (1Tabor H. Stetten Jr., D. Carrigan W.T. NIH: An Account of Research in Its Laboratories and Clinics. Academic Press, New York1984: 220-229Google Scholar). At the time NIH had little prestige compared with academic institutions. What it did offer was regular support and a great deal of scientific freedom. Leon Heppel, Arthur Kornberg, Herbert Tabor, and Bernard Horecker used part of their freedom to learn biochemistry. Horecker, already an experienced enzymologist, helped them find their way. Their primary educational tool was a private, daily, lunchtime journal club; according to legend, the only day off was Christmas. Kornberg was the dominant personality. Years later, long after he had left the NIH and the club was open to other colleagues and even postdoctoral fellows, any proposed change in the lunch club format elicited the question: “What would Arthur think?” The biochemistry of phosphate-containing compounds became a central interest of several in the original lunch club group. Horecker's work led to a description of the pentose phosphate pathway (2Horecker B.L. The pentose phosphate pathway..J. Biol. Chem. 2002; 277: 47965-47971Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Kornberg began working on the enzymatic synthesis of pyrophosphate, coenzymes, and nucleotides, and he has told the story about how these investigations led to the discovery of DNA polymerase I in the three years after he left the NIH for Washington University in 1953 (1Tabor H. Stetten Jr., D. Carrigan W.T. NIH: An Account of Research in Its Laboratories and Clinics. Academic Press, New York1984: 220-229Google Scholar, 3Kornberg A. For the Love of Enzymes. Harvard University Press, Cambridge, MA1989: 121-169Google Scholar). Heppel (Fig. 1) had earned a Ph.D. in biochemistry from the University of California at Berkeley in 1937 and an M.D. in 1941 from the University of Rochester. After completing a medical internship at Strong Memorial in Rochester, he carried out toxicology research at the NIH during the war years. By 1950 he, together with his long time colleague Russell J. Hilmoe, had begun experiments on enzymes that catalyze the hydrolysis and phosphorolysis of polyribonucleotides and their derivatives. They studied 5′-nucleotidase (4Heppel L.A. Hilmoe R.J. Purification and properties of 5′-nucleotidase..J. Biol. Chem. 1951; 188: 665-676Abstract Full Text PDF PubMed Google Scholar), inorganic pyrophosphatase (5Heppel L.A. Hilmoe R.J. Purification of yeast inorganic pyrophosphatase..J. Biol. Chem. 1951; 192: 87-94Abstract Full Text PDF PubMed Google Scholar), and the hydrolysis and phosphorolysis of purine ribosides and ATP (6Heppel L.A. Hilmoe R.J. Phosphorolysis and hydrolysis of purine ribosides by enzymes from yeast..J. Biol. Chem. 1952; 198: 683-694Abstract Full Text PDF PubMed Google Scholar, 7Heppel L.A. Hilmoe R.J. Mechanism of enzymatic hydrolysis of adenosine triphosphate..J. Biol. Chem. 1953; 202: 217-226Abstract Full Text PDF PubMed Google Scholar). One of their main interests was a phosphodiesterase found in bovine spleen (8Heppel L.A. Hilmoe R.J. Spleen and intestinal phosphodiesterases..Methods Enzymol. 1955; 2: 565-570Crossref Scopus (9) Google Scholar). Although they purified the enzyme to some extent, they were unable to characterize satisfactorily the products of digestion of polynucleotides. Heppel, then just over 40 years old, decided to take a leave from NIH and spend 1953 in Roy Markham's laboratory in Cambridge, England, where he hoped to solve the problem of the structure of the products. Markham could teach Heppel his innovative techniques for partition chromatography on paper strips as well as pioneering paper electrophoresis methods for nucleic acid components. With the support of a Guggenheim Fellowship and a travel grant from the American Cancer Society, Heppel, his wife Adelaide, and their two sons (ages 5 and 1) sailed across the Atlantic on the S.S. United States. Markham, a few years younger than Heppel, was at the Molteno Institute in the Plant Virus Research Unit of the Agricultural Research Council. Then, as now, the name of a research department need not reflect the most exciting ongoing science. Markham and his associates were mainly concerned with nailing down the structure of the internucleotide bonds in RNA. Did they go from a 5′-hydroxyl to a 2′-hydroxyl on the ribose moiety or, in analogy with DNA, to a 3′-hydroxyl? Both acid and alkaline hydrolysis yielded 2′,3′-cyclic phosphodiester intermediates and a mixture of 2′- and 3′-mononucleotides as final products. Similarly, RNase A digestion went through the 2′,3′-cyclic intermediates although it eventually gave only 3′-mononucleotides. Neither of these methods could unequivocally establish which kind of bond was in the RNA itself. The analogy with the 5′,3′ bond in DNA and the fact that RNase A hydrolyzed pyrimidine 3′-benzyl but not pyrimidine 2′-benzyl phosphodiester favored the 3′-hydroxyl link, at least for pyrimidine nucleotides in RNA (9Brown D.M. Todd A.R. Nucleotides. Part XXI. The action of ribonuclease on simple esters of the monoribonucleotides..J. Chem. Soc. 1953; : 2040-2049Crossref Scopus (22) Google Scholar). However, the standard of proof required by biochemists of the day demanded a more direct demonstration. The primary tool at hand was ingenious use of highly specific enzymes. Thus, a specific phosphodiesterase from snake venom that yielded 5′-mononucleotides from RNA had established the link on one side of the internucleotide bond (10Cohn W.E. Volkin E. On the structure of ribonucleic acids. I. Degradation with snake venom diesterase and the isolation of pyrimidine diphosphates..J. Biol. Chem. 1953; 203: 319-332Abstract Full Text PDF PubMed Google Scholar). By April of 1953, Markham's laboratory with Heppel's help had settled the issue; the bond was 5′ to 3′. Markham and P. R. Whitfeld, an Australian postdoctoral fellow, reported that when the dinucleotide monophosphates GpC, GpU, ApC, and ApU (separated from RNase A digests after dephosphorylation) were oxidized to dialdehydes by periodate (with cleavage between the 2′- and 3′-hydroxyls of the pyrimidine ribose) and treated at pH 10, 3′-GMP and 3′-AMP were produced (11Whitfeld P.R. Markham R. Natural configuration of the purine nucleotides in ribonucleic acids..Nature. 1953; 171: 1151-1152Crossref PubMed Scopus (34) Google Scholar). In the accompanying paper, Heppel, along with Markham and Hilmoe, showed that the partly fractionated phosphodiesterase from bovine spleen that he had brought to England yielded 3′-purine mononucleotides from compounds such as ApApU with no intermediary formation of cyclic nucleotides (8Heppel L.A. Hilmoe R.J. Spleen and intestinal phosphodiesterases..Methods Enzymol. 1955; 2: 565-570Crossref Scopus (9) Google Scholar, 12Heppel L.A. Markham R. Hilmoe R.J. Enzymatic splitting of purine internucleotide linkages..Nature. 1953; 171: 1152Crossref PubMed Scopus (2) Google Scholar, 13Whitfeld P.R. Heppel L.A. Markham R. The enzymic hydrolysis of ribonucleoside 2′,3′-phosphates..Biochem. J. 1955; 60: 15-19Crossref PubMed Scopus (31) Google Scholar). Further, the enzyme could not hydrolyze purine 2′-benzyl phosphodiesters (14Brown D.M. Heppel L.A. Hilmoe R.J. Nucleotides. Part XXIV. The action of some nucleases on simple esters of monoribonucleotides..J. Chem. Soc. 1954; : 40-46Crossref Google Scholar). Heppel worked as prodigiously in England as he did at home. And as at home, he and Adelaide enjoyed the sites and life around them despite the difficulties of living with two small children in postwar England. Food rationing was still in place, and the standard of living was not what they had left behind in the United States. Careful and sensitive observers, the Heppels could make a short walk or a single painting into a world of experience. Besides pinning down the internucleotide bond in RNA, Heppel that year also demonstrated that RNase A and the spleen phosphodiesterase could catalyze nucleotide transfer reactions; for example, incubation of 2′,3′-cyclic AMP, methanol, and enzyme yielded adenosine 3′-methyl phosphodiester (15Heppel L.A. Whitfield P.R. Synthesis and interconversion of simple esters of ribomononucleotides..Biochem. J. 1954; 60: 1-7Crossref Scopus (7) Google Scholar). RNase A would even use a nucleoside or nucleotide as acceptor and catalyze the synthesis of polyribonucleotides (16Heppel L.A. Whitfield P.R. Markham R. Synthesis of polynucleotides..Biochem. J. 1954; 60: 8-15Crossref Scopus (22) Google Scholar). In this paper, the authors proposed a modification of the abbreviations for polynucleotides then in use, and these became the conventions that we use to this day (e.g. putting the 5′-end at the left). Although it soon became clear that polyribonucleotides are not synthesized by transphosphorylation (or transnucleotidation) in cells, the work had several consequences. Most importantly, when Severo Ochoa and Marianne Grunberg Manago discovered polynucleotide phosphorylase (PNPase) in extracts of Azotobacter vinelandii, they turned to Heppel for collaboration in characterizing the polymer products. More personally, it was the work on transphosphorylation that attracted the interest of Joseph S. Fruton, my Ph.D. professor at Yale, and led him to recommend that I apply to Heppel for postdoctoral training. By the time I joined Heppel's NIH laboratory in October of 1956 he was deeply involved in analyzing the polyribonucleotides formed from nucleoside 5′-diphosphates by action of PNPase (17Heppel L.A. Ortiz P.J. Ochoa S. Studies on polynucleotides synthesized by polynucleotide phosphorylase..J. Biol. Chem. 1957; 229 (695–710): 679-694Abstract Full Text PDF PubMed Google Scholar). Earlier that year, he and Ochoa (18Ochoa S. Heppel L.A. Polynucleotide synthesis.in: McElroy W.D. Glass B. The Chemical Basis of Heredity. The Johns Hopkins University Press, Baltimore, MD1957: 615-638Google Scholar) had summarized their results at a conference where they also considered whether PNPase could be responsible for RNA synthesis and speculated on possible mechanisms of DNA synthesis. Arthur Kornberg presented some of the early experiments on DNA polymerase I of Escherichia coli at the same meeting. It is easy to imagine the excitement in Baltimore during that symposium as participants heard the first clues to the synthesis of polynucleotides. These discoveries opened a new era for biochemistry, although as it turned out PNPase was not responsible for RNA synthesis and DNA polymerase I was not the key enzyme involved in DNA replication. Meanwhile, Heppel was becoming interested in the mechanisms of the PNPase polymerization reaction as well as of the reverse reaction, the phosphorolysis of the polymers. These two areas became the focus of my own research when I joined Heppel's laboratory. In the next few years others at the NIH, notably Dan Bradley, David Davies, Gary Felsenfeld, Marie Lipsett, Todd Miles, Alex Rich, and later Martin Gellert used the polymers produced by PNPase action to study the physical properties of long polyribonucleotides. At this time, very few biochemists worked with RNA or polyribonucleotides or indeed with DNA. Two brief sections in Volume II of Methods in Enzymology, published in 1955, were sufficient to deal with the known enzymes of phosphate and nucleic acid metabolism; Kornberg and Heppel together contributed a significant percentage of the papers. The community was not even large enough to have a Gordon Conference to call its own. Through most of the 1950s, a single annual conference was entitled “Proteins and Nucleic Acids.” Then, in 1959, the Gordon Conference organization announced that because the scope of research in both areas had become “so wide” each topic would have its own conference, although only in alternating years. The 1960 conference, still entitled “Proteins and Nucleic Acids,” was the first devoted exclusively to nucleic acids. Finally, in 1962, there was a Gordon Conference on “Nucleic Acids” co-chaired by Heppel and Cyrus Levinthal. In 1958, when Horecker left the NIH, Heppel succeeded him as chief of the Laboratory of Biochemistry and Metabolism, National Institute of Arthritis and Metabolic Diseases. Before he left, Horecker changed my status from postdoctoral fellow to regular employment as a research chemist. This was a notable appointment because it demonstrated that, in contrast to standard practices in university faculties, Horecker, Heppel, and indeed the NIH provided opportunities for women to become independent investigators. Heppel's new responsibilities never slowed his laboratory work. He set a remarkable tone for the group of researchers under his general oversight. He was “careful, meticulous, childish, and screwy, but always calculatedly so” (19Martin R.G. A revisionist view of the breaking of the genetic code.in: Stetten Jr., D. Carrigan W.T. NIH: An Account of Research in Its Laboratories and Clinics. Academic Press, New York1984: 281-296Crossref Google Scholar), and he was also enormously supportive of the staff including most especially the postdoctoral fellows. Audrey Stevens, who came because she wanted to learn about RNA from Heppel, flourished in the laboratory. In 1960 she was one of the three investigators who simultaneously discovered RNA polymerase. Her paper, like others written by Heppel's postdoctoral fellows, is published under her name alone (20Stevens A. Incorporation of the adenine ribonucleotide into RNA by cell fractions from E. coli..Biochem. Biophys. Res. Commun. 1960; 3: 92-96Crossref Scopus (28) Google Scholar). She recalls that unlike today's laboratory heads he “worked in the lab every day and did not mind someone looking over his shoulder.” He too looked over shoulders peering at the protocol attached to a clipboard propped up on the bench. Custom required that the clipboard contain two sheets of loose-leaf paper separated by carbon paper so that both Heppel and the postdoctoral fellow would have copies; copying machines were yet to be invented. Often, he suggested that the carefully planned protocol be changed just as the experiment was being set up. The only excuse for this irksome behavior was that he was usually correct. Heppel's laboratory had become a magnet for the growing number of investigators interested in learning how to work with polyribonucleotides and RNA. They were attracted by his store of specific enzymes and knowledge of paper electrophoretic and chromatographic methods as well as the spectrophotometric techniques used to analyze the products of nucleic acid synthesis and degradation. They were also attracted by Heppel's generous and selfless cooperation and hospitality. They knew that their time would be efficiently spent as he scurried around days and evenings helping them at the same time that he kept up with his own experiments. Heppel's visits to Kornberg and his colleagues in St. Louis and to Gobind Khorana in Vancouver his as what be a one with a well of and a huge store of from laboratory were and Marianne Grunberg Manago came for several after she had to Gobind Khorana too visits to learn nucleic acid who had discovered PNPase E. a postdoctoral fellow in laboratory in St. came to work with Heppel on enzyme and of the polymer when to the Institute he from Heppel, a of enzymes with which to his work. I. R. that when he the NIH laboratory for in 1958, Heppel his days from to was but he had how to on by the Heppel and Herbert had postdoctoral fellows, and to into the by each for the journal I for years and through the club kept up with the of new discoveries a of for my also in Heppel began these days with and even from an and he was as as any of to be the of the as but at least was the to clear up Heppel's was where he his opened and when the was too to he it all with a of paper and began a new This behavior was the one in Heppel's such as from to laboratory every and putting some between his and used or On one had to through the and of What he did with the was never On one he down the after a from and the on the he found one he had from David was to and to The was the of nucleotide with the cyclic that had after ATP with story is told in I. PubMed Scopus Google Scholar). the 1950s, studies on nucleic acid biochemistry and were still The two to one and when they of of and were because biochemists the were too to and the the biochemists had no for The of Press, New for a description of the as for a as did after he and in 1941 that a a P. An Press, New Scholar). Heppel, who was on his never the or even one or It is that in the group knew about that the lunch club heard about and the of However, I not of the about or RNA that were in the did about the work of Volkin and that after with E. coli synthesized an RNA with base composition to that of the E. of ribonucleic acid after of Escherichia 2: PubMed Scopus Google Scholar). we the to establish a for synthesis especially after NIH colleague began with these came the day when from Heppel's synthesized with was used by and his colleague in the experiment that the genetic for By the of we knew a about and RNA and several of were at work polyribonucleotides with known of two or more bases for the experiments on the other These two discoveries by RNA in and the of the genetic that and the new biochemistry were working on the same In Heppel left the NIH to become professor of biochemistry at By this time his research had to the of the of enzymes in E. At he began studying the of cell to work that into an interest in of He also his of to colleagues left The up on and his about and they that the to his for example, he is the name of the in The he that was him that a in the the next day in the laboratory was more than years but in fact it was mainly It is only in the few years that Heppel his in the laboratory.

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