The Discovery of RNA Polymerase
2005; Elsevier BV; Volume: 280; Issue: 52 Linguagem: Inglês
10.1074/jbc.x500006200
ISSN1083-351X
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoIn all organisms, genes coding for proteins are transcribed by the multisubunit complex, DNA-dependent RNA polymerase. In prokaryotes, this is accomplished by the core RNA polymerase linked to a number of sigma factors that specify its binding to different promoter elements. In eukaryotes, this process is more complex because of the presence of three distinct RNA polymerases, each responsible for the transcription of a different class of RNA. During the past few years, structural and biochemical studies have provided enormous insight into how these macromolecular machines interact with DNA and carry out many of the detailed steps in the initiation and elongation of RNA chains (1Murakami K.S. Darst S.A. Curr. Opin. Struct. Biol. 2003; 13: 31-39Crossref PubMed Scopus (381) Google Scholar, 2Boeger H. Burshnell Davis R. Griesenbeck J. Lorch Y. Strattin J.S. Westover K.D. Kornberg R.D. FEBS Lett. 2005; 579: 899-903Crossref PubMed Scopus (100) Google Scholar). Today, DNA microarray experiments permit the simultaneous analysis of the transcription of thousands of genes in a single experiment under a variety of conditions. Because of the availability of data banks containing sequences of many genomes, we can change or delete promoter sites selectively to alter transcription. More than 50 years ago such techniques and knowledge were unimaginable. However, it was during the period spanning 1955–1961 that discoveries were made which resulted in the isolation of polynucleotides phosphorylase, Escherichia coli DNA polymerase I, and DNA-dependent RNA polymerase and the solution of the genetic code, all of which contributed importantly to the future of research. Recollections concerning the discovery of polynucleotide phosphorylase (3Grunberg-Manago M. Biochim. Biophys. Acta. 1989; 1000: 59-64Crossref PubMed Scopus (2) Google Scholar), DNA polymerase I (4Lehman I.R. J. Biol. Chem. 2003; 278: 34733-34738Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), and a historical review of the deciphering of the genetic code (5Nirenberg M. Trends Biochem. Sci. 2004; 29: 46-54Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) have appeared. The purpose of this article is to describe the discovery of DNA-dependent RNA polymerase. Because it is included among articles entitled “Reflections,” I have taken the liberty to describe this discovery as a personal journey and how I and others contributed to this important finding.Early Training in BiochemistryMy early training was carried out in the Department of Biochemistry at Western Reserve University where I received my Ph.D. in 1953. The biochemistry department at that time was chaired by Harland G. Wood. The focus of the research effort of the department was primarily intermediary metabolism. Though all graduate students were required to take advanced chemistry, biochemistry, and microbiology courses, my exposure to what could be described as the beginnings of molecular biology was minimal and my appreciation of genetics was rudimentary at best. These deficiencies were personally troubling because my sister Zella was married to Salvador Luria. Dating back to 1945 and my first visit to the Cold Spring Harbor Biological laboratories where the Lurias were spending a sabbatical year, I had listened with little comprehension to Salva, Max Delbrück, Al Hershey, Renato Dulbecco, and other luminaries of the phage field discuss their research. During my early graduate training I noticed their disdain for biochemistry, and years later, I was more than pleased when they contacted me for biochemical advice. In 1951, hoping to overcome this lack of knowledge, I took the phage course at Cold Spring Harbor. I recall being excited about the course and the phage field, but this enthusiasm was dissipated quickly on my return to Cleveland. My fellow students, who at that time included Sasha Englard, Paul Berg, and Harry Rudney, among others, were too engrossed in their own research efforts to listen to my description of the newly discovered area of lysogeny or my appreciation of the Luria-Delbrück fluctuation experiment. I recall Luria telling me during a visit to Urbana in the summer of 1952 that Jim Watson (who had been a graduate student in his laboratory at Indiana University) was carrying out important work on the structure of DNA. It made little impression on me since by that time I was immersed in studying the enzymatic phosphorylation of vitamin B6 derivatives and their function as coenzymes. The excellent training we graduate students received in biochemistry at Western Reserve was influenced by Harland Wood's work ethic and devotion to science. His extraordinary research accomplishments and role in training young scientists have been chronicled in wonderful articles by David Goldthwait and Richard Hanson (6Goldthwait D.A. Hanson R.W. Biogr. Mem. Natl. Acad. Sci. 1996; 69: 3-36Google Scholar) and Paul Berg (7Berg P. J. Biol. Chem. 2003; 278: 40417-40424Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Wood invited many distinguished scientists of the period who presented exciting seminars that kept us up to date with their new discoveries. These included Hans Krebs, Fritz Lipmann, Ephraim Racker, Severo Ochoa, Fred Sanger, Arthur Kornberg, Seymour Cohen, Melvin Calvin, and Bernard Horecker. Because a substantial focus of the department was on CO2 fixation, I became interested in photosynthesis. I was particularly excited by the seminars given by Calvin and Horecker. Between 1950 and 1952, Calvin's group carried out in vivo studies on CO2 fixation in photosynthetic organisms while Horecker's laboratory examined the enzymes involved in the oxidation of 6-phosphogluconate (Warburg-Dickens-Lipmann pathway) in animal tissues and yeast. Independently, they both identified a number of key common phosphorylated sugar derivatives (ribulose phosphate in particular), suggesting that the pathway by which CO2 fixation occurred in photosynthesis was biochemically tractable. I thought that the purification and isolation of the enzymes involved in photosynthetic CO2 fixation was the best way to define this pathway. Because this was the approach Horecker's group used to elucidate the oxidation of 6-phosphogluconate, which then led to the discovery of the transketolase and transaldolase, I decided to apply for postdoctoral training in his laboratory at the National Institutes of Health. Horecker graciously accepted me as a postdoctoral fellow. In the interim period, Wood convinced me that I needed additional training in chemistry. For that reason, immediately after receiving my Ph.D. degree, I spent 1953–1954 working in Albert Neuberger's laboratory at the Institute for Medical Research at Mill Hill in London, England, where I carried out stereochemical organic syntheses. I was given the problem of synthesizing hydroxyornithine in order to evaluate whether it rather than hydroxyproline was a constituent of collagen. Though I never accomplished this feat, I did synthesize both d- and l-carnitine primarily because they could be readily made from d- and l-isoserine, well defined stereochemical intermediates I had prepared for the production of hydroxyornithine. I never published this work because I naively considered these compounds biologically uninteresting at the time. Shortly thereafter, carnitine was shown to function as a carrier of acetyl groups through the mitochondrial membrane (8Friedman S. Fraenkel G. Arch. Biochem. Biophys. 1955; 59: 491-501Crossref PubMed Scopus (75) Google Scholar, 9Bremer J. J. Biol. Chem. 1962; 237: 3628-3632Abstract Full Text PDF PubMed Google Scholar). However, this period did give me the opportunity to learn some organic chemistry, how to use the many volumes of Beilstein, and travel.Postdoctoral Training at the NIHBetween 1954 and 1956, I was a postdoctoral fellow at the National Institutes of Health in Horecker's laboratory. During this period, in collaboration with Bernie, Pauline (Polly) Smyrniotis, and Arthur Weissbach, we isolated and characterized a number of enzymes and substrates that firmly established the photosynthetic CO2 fixation cycle proposed by Calvin, which Horecker has summarized in a “Reflections” article (10Horecker B.L. J. Biol. Chem. 2002; 237: 47965-47971Abstract Full Text Full Text PDF Scopus (113) Google Scholar). Horecker, Ed Heath, and I also solved a vexing biochemical puzzle at that time, which involved the fermentation of pentoses by several Lactobacillus species whereby the methyl and carboxyl residues of acetate arise from the carbon atoms 1 and 2 of pentose, respectively, whereas lactate is derived from carbon atoms 3, 4, and 5. We demonstrated that xylulose 5-phosphate was cleaved phosphorolytically to acetyl phosphate and triose phosphate by a thiamine pyrophosphate-dependent enzyme that we called phosphoketolase (11Heath E.C. Hurwitz J. Horecker B.L. J. Am. Chem. Soc. 1956; 78: 5494Crossref Google Scholar). This highly productive period was due largely to Horecker's impressive expertise in carbohydrate metabolism, enthusiasm for research, and general positive personality. Harry Eagle, also cognizant of this buoyant demeanor, aptly described Bernie as a reincarnated Dr. Pangloss. The exploits of the Enzyme Section, first organized by Arthur Kornberg and headed by Horecker during the mid-1950s, included a daily luncheon seminar on literature (and any new exciting findings) with Herbert Tabor, Celia Tabor, Alan Mehler, Leon Heppel, Herman Kalckar, Jesse Rabinowitz, Bruce Ames, and anyone else willing to squeeze into the small library of Building 3 or later on the 9th floor of the Clinical Center and have been described in a number of “Reflections” articles (10Horecker B.L. J. Biol. Chem. 2002; 237: 47965-47971Abstract Full Text Full Text PDF Scopus (113) Google Scholar, 12Singer M. J. Biol. Chem. 2003; 278: 47351-47356Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). During this period, I recall Severo Ochoa's visit at which time he was scheduled to present a seminar on aspects of oxidative phosphorylation in Azotobacter vinelandii. Though he initiated his seminar discussing β-hydroxybutyrate oxidation, I recall being tremendously excited when he described the experiments that he and Marianne Grunberg-Manago had carried out, which led to the discovery of polynucleotide phosphorylase (PNPase). At that time, Ochoa elicited Leon Heppel's expertise to help characterize the polymer products. Leon, in collaboration with Roy Markham's group at the Molteno Institute in Cambridge, England, pioneered many of the chemical and enzymological techniques that established the 5′-3′ internucleotidic phosphodiester linkage in RNA. During his studies with the A. vinelandii PNPase, Heppel noted that the preparations were contaminated with an activity that converted ADP to ribose phosphate and adenine, which severely limited the synthesis of poly(A). He proposed that Horecker, he, and I jointly investigate this problem. We quickly showed that it was due to an enzyme that cleaved AMP to adenine and ribose 5-phosphate. The PNPase preparations also contained a myokinase activity that converted ADP to AMP and ATP. Surprisingly, the cleavage of AMP required ATP only as an effector (13Hurwitz J. Heppel L.A. Horecker B.L. J. Biol. Chem. 1957; 226: 525-540Abstract Full Text PDF PubMed Google Scholar). During these studies, I delighted in my interactions with Leon. These included his many handwritten notes (sometime 5–10 pages), which often included totally “off the wall” quotes from newspapers, information about various classical symphonies, and quizzes. During our work on the AMP-cleaving activity, he once appeared in the doorway of my laboratory in Building 10 asking what he should do next. When I responded that I thought that he should think more about what we were doing, he quickly stated “I don't have the time. Just tell me what to do.” I suspect that these pranks provided Heppel periods of respite from his focused and dedicated research efforts that resulted in his many important contributions to RNA biochemistry, already described by Maxine Singer (12Singer M. J. Biol. Chem. 2003; 278: 47351-47356Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar).Early Years in St. Louis and Studies in Nucleic Acid BiochemistryThough I thoroughly enjoyed my postdoctoral period at the NIH, in 1956 I joined the Microbiology Department of Washington University in St. Louis, MO, which was chaired by Arthur Kornberg. My decision to leave the NIH was influenced in part by my strong aversion to the loyalty oath required at government institutions and more so by my desire to return to a more academic environment and interests in studying nucleic acid biochemistry. Kornberg's research accomplishments were already legendary, and the department he had assembled in St. Louis was first rate and included Paul Berg, Melvin Cohn, David Hogness, and Dale Kaiser. At that time, I. R. (Bob) Lehman was a postdoctoral fellow in Kornberg's laboratory and was appointed to the faculty soon after. The group was highly congenial, mutually supportive, and intensely research oriented. The department was housed on the fourth floor of an ancient clinic building that bustled with the influx and efflux of many patients. A rickety old caged elevator carried us to our laboratories on the fourth floor. As the elevator ascended the patients milling about in the waiting rooms became more visible because of the high ceilings of the large waiting rooms. On one occasion while riding in the elevator with Kornberg, he told me that he felt exhilarated by the clinic because it kept him in touch with reality. Years later, I told him that I was impressed by his positive attitude about the old clinic building at Washington University. He looked at me with horror and said, “I thought it was terrible.”During my postdoctoral period at the NIH and our interests in pentose metabolism, Horecker and I wondered how deoxyribose was formed but made no effort to investigate this problem. Earlier studies by Ephraim Racker (14Racker E. J. Biol. Chem. 1952; 196: 347-365Abstract Full Text PDF PubMed Google Scholar) demonstrated that deoxyribose 5-phosphate could be formed by an aldol condensation of glyceraldehyde 3-phosphate and acetaldehyde, a reaction catalyzed by the enzyme deoxyribose-phosphate aldolase. This enzyme was found widely distributed in microorganisms and animal tissues. However, its physiological role as the source of deoxyribose in DNA was questioned by the findings of I. A. Rose and B. S. Schweigert (15Rose I.A. Schweigert B.S. J. Biol. Chem. 1953; 202: 635-645Abstract Full Text PDF PubMed Google Scholar), who demonstrated that uniformly labeled ribonucleosides (isolated from Euglena grown on 14CO2) were converted into deoxynucleotides of DNA in vivo with no change in the ratio of the radioactivity found in the base and sugar. Impressively, these results were obtained with cytidine in E. coli and the rat as well was with GMP and CMP in Lactobacillus leichmanii. I decided to study whether ribonucleotides were reduced directly to deoxynucleotides when I got to St. Louis.However, soon after I arrived in St. Louis, Harland Wood contacted me and wanted to know whether the glucose fermentation pathway used by Leuconostoc mesenteroides included the phosphorolytic cleavage of pentose that Horecker, Heath, and I had discovered. His laboratory had successfully used the pattern of C14 in the glucose of liver glycogen as an indicator of carbohydrate metabolic pathways (16Bernstein I.A. Lentz K. Malm M. Schambye P. Wood H.G. J. Biol. Chem. 1955; 215: 137-152Abstract Full Text PDF PubMed Google Scholar). They showed that glucose fermentation by L. mesenteroides produced equimolar quantities of CO2, ethanol, and lactate from the carbon atoms 1, 2 and 3 and 4–6 of glucose, respectively. Because the reagents required to detect phosphoketolase were readily available (albeit in Horecker's freezers at the NIH), I had little difficulty in showing that xylulose 5-phosphate was phosphorolyzed by extracts of L. mesenteroides to acetate and triose phosphate (17Hurwitz J. Biochim. Biophys. Acta. 1958; 28: 599-602Crossref PubMed Scopus (25) Google Scholar), verifying the fermentation pathway which made the organism such a useful means for the isolation of each glucose carbon atom. Upon completion of this diversion, I began studies on the reduction of ribonucleotides to deoxynucleotides. At this time, a highly sensitive colorimetric assay for deoxyribose appeared (16Bernstein I.A. Lentz K. Malm M. Schambye P. Wood H.G. J. Biol. Chem. 1955; 215: 137-152Abstract Full Text PDF PubMed Google Scholar). It depended on the periodate oxidation of the sugar to malondialdehyde, which on interaction with thiobarbituric acid yielded a chromogen with an absorption maximum at 532 mμ. Incubation of AMP or ribose 5-phosphate with extracts of E. coli resulted in substantial levels of the chromogen. However, the spectrum of the product formed in the periodate-thiobarbituric acid test differed from that found with deoxyribose. Arthur Weissbach, who was then working at the NIH, made similar observations, and we combined our efforts to identify the product. In a short time, using chemical and enzymatic approaches, we showed the product was 2-keto-3-deoxyheptonic acid, an intermediate in shikimic acid formation, which was formed by the enzymatic interaction between erythrose 4-phosphate and phosphoenolpyruvate, products readily produced from ribose 5-phosphate (19Weissbach A. Hurwitz J. J. Biol. Chem. 1959; 234: 705-709Abstract Full Text PDF PubMed Google Scholar, 20Hurwitz J. Weissbach A. J. Biol. Chem. 1959; 234: 710-712Abstract Full Text PDF PubMed Google Scholar).I then used labeled nucleotides to explore the conversion of ribonucleotides to deoxynucleotides. Because labeled ribonucleotides of high specific activity were not commercially available at the time, I isolated this material initially by growing E. coli in the presence of large amounts of 32P in low phosphate media and then degrading the 32P-labeled RNA with venom phosphodiesterase to 5′-mononucleotides, which were then chromatographically separated on Dowex 1-Cl columns. Contaminating deoxynucleotides were removed by paper chromatography in borate-containing solvents. I replaced this laborious procedure with one suggested by Gobind Khorana. At that time, Khorana was at the University of British Columbia in Canada and visited St. Louis to complete studies that he and Kornberg were carrying out on the structure of phosphorylribose pyrophosphate (PRPP), the active ribose phosphate derivative involved in the synthesis of pyrimidine and purine nucleotides (21Khorana H.G. Fernandez J.F. Kornberg A. J. Biol. Chem. 1958; 230: 941-948Abstract Full Text PDF PubMed Google Scholar). While in St. Louis, Gobind worked in my laboratory during which time we became good friends. He suggested a simple chemical procedure that his group had developed. For this purpose, 32Pi was converted to polyphosphoric acid at 250 °C, neutralized, and then incubated with cytidine at 60 °C for 3–4 h. This resulted in the formation of a mixture of labeled 2′-, 3′-, and 5′-CMP from which 5′-CMP could be isolated. I then found that the incubation of 32P-labeled 5′-CMP with crude extracts of E. coli resulted in the production of low levels of 32P-labeled 5′-dCMP, which were resolved from 32P-labeled 5′-CMP by borate-paper chromatography. I noted that the reaction required ATP and that treatment of reaction mixtures with potato apyrase (which cleaved the β and γ phosphate residues of NTPs) increased the yield of [32P]dCMP. My next task was to purify the system and identify the reducing agent. At this point, I made a fateful decision to change the paper chromatographic assay for a more rapid one. Kornberg's laboratory (which at that time included Maurice Bessman, Bob Lehman, and Ernie Simms) was examining the action of E. coli DNA polymerase I, first discovered by Arthur's group in 1955. Their exquisite work had shown that purified enzyme fractions incorporated all 4 dNTPs into DNA and (most critical for the assay I planned to use) worked specifically with dNTPs and not rNTPs. I decided to couple the [32P]CMP conversion to [32P]dCMP reaction with the DNA polymerase I system and measure the incorporation of dCMP into DNA. Acid precipitation was a more rapid means to measure this reaction. For this purpose, I incubated [32P]CMP with crude extracts of E. coli (which contained kinases capable of forming the nucleoside di- and triphosphate derivatives) and then added dATP, dGTP, dTTP, and purified DNA polymerase I. Indeed labeled DNA accumulated. The reaction required crude extracts, the dNTPs, and DNA and was blocked by the addition of pancreatic DNase. Initial attempts to further purify the components necessary to support the incorporation of 5′-32P-labeled CMP into DNA resulted in considerable loss of activity. Searching for factors that increased the yield, I discovered that a mixture of Mn2+ + Mg2+ was considerably more effective than Mg2+ alone. Much to my surprise, when I isolated the labeled DNA products formed in the reaction and degraded this material with pancreatic DNase I plus venom phosphodiesterase, conditions that I expected would yield 32P-labeled 5′-dCMP, I observed that the labeled CMP had been incorporated directly into DNA with no detectable labeled dCMP present in DNA. The 32P-labeled polynucleotide product was rendered partially acid-soluble after either RNase or NaOH hydrolysis but completely acid-soluble by DNase I digestion. These properties suggested that rCMP had been incorporated distributively throughout the polynucleotide chain synthesized. Reactions that were carried out with [α-32P]CTP + UTP + GTP +ATP (no dNTPs) yielded low levels of labeled acid-insoluble material, which were reduced upon omission of a single rNTP. I published this work in a paper entitled “The Enzymatic Incorporation of Ribonucleotides in Polydeoxynucleotide Material” in which I indicated that the biological significance of this enzymatic reaction was unclear (22Hurwitz J. J. Biol. Chem. 1959; 234: 2351-2358Abstract Full Text PDF PubMed Google Scholar). Nearly 5 years later, it was shown that the presence of Mn2+ altered the nucleotide specificity of DNA polymerase I, leading to the incorporation of both rNTPs and dNTPs into DNA (23Berg P. Fancher H. Chamberlin M. Vogel H. Bryson B. Lampen J.O. Informational Macromolecules. Academic Press, New York1963: 469Google Scholar). In retrospect, it is likely that my findings were due to multiple activities including DNA polymerase I and possibly low levels of DNA-dependent RNA polymerase. How I missed discovering RNA polymerase during these studies in 1958 mystifies me to this day. Though my initial goal was to discover how deoxynucleotides were formed from ribonucleotides, I never returned to this problem. In 1961, Peter Reichard's laboratory demonstrated that rCDP was converted to dCDP by extracts of E. coli (24Reichard P. Baldesten A. Rutberg L. J. Biol. Chem. 1961; 236: 1150-1157Abstract Full Text PDF PubMed Google Scholar) and later showed that the reducing agent was a pair of intrinsic sulfhydryl groups on the enzyme (ribonucleotide reductase) that was regenerated after catalyzing the reduction by thioredoxin, which acts as a protein disulfide reductase (25Reichard P. The Biosynthesis of Deoxyribose Ciba Lectures in Biochemistry. John Wiley & Sons, Inc., New York1967Google Scholar).The Discovery of DNA-dependent RNA PolymeraseIn 1958, I moved to New York and joined the Microbiology Department at New York University, School of Medicine. Bernie Horecker was appointed chairman of the department. At that time, influenced by the work I carried out in St. Louis, I decided to focus on RNA synthesis. During this period, our thinking about the biosynthesis of RNA was dominated by the action of PNPase first discovered by Grunberg-Manago and Ochoa (3Grunberg-Manago M. Biochim. Biophys. Acta. 1989; 1000: 59-64Crossref PubMed Scopus (2) Google Scholar) and independently somewhat later by Uriel Littauer when he was a postdoctoral fellow in Arthur Kornberg's laboratory (26Littauer U.Z. Kornberg A. J. Biol. Chem. 1957; 226: 1077-1092Abstract Full Text PDF PubMed Google Scholar). The role of PNPase in synthesizing biologically relevant RNA, however, was questioned by its mode of action. The enzyme required high levels of ribonucleoside diphosphates as substrate and produced Pi and high molecular weight RNA polymers whose composition depended on the level and particular nucleoside diphosphates added to the reaction. The reaction was freely reversible, and RNA polymers were readily phosphorolyzed to the corresponding ribonucleoside diphosphates. There was no evidence of a template-directed synthesis of RNA, though at the time of its discovery the possibility was raised that the aberrant products formed resulted from the loss of factors during its purification that were required to form more physiological RNA products. Though further studies validated these concerns and PNPase was found to be a variable component of the prokaryotic degradosome, which regulates RNA hydrolysis, the enzyme proved to be of enormous importance. It provided the means for the synthesis of many different RNAs that helped to define the genetic code and the first hybridization experiments between poly(A) and poly(U), carried out by the laboratories of Alex Rich and Robert Warner (27Rich A. Davies D.R. J. Am. Chem. Soc. 1956; 78: 3548Crossref Scopus (52) Google Scholar, 28Warner R.C. J. Biol. Chem. 1957; 229: 711-724Abstract Full Text PDF PubMed Google Scholar) and between RNA and DNA (poly(A) and a synthetic poly(dT) oligomer chemically synthesized by Khorana) by Alex Rich (29Rich A. Proc. Natl. Acad. Sci. U. S. A. 1960; 46: 1044-1053Crossref PubMed Google Scholar). Marmur, Doty, and their colleagues also showed that complementary single-stranded DNAs formed by heat denaturation of duplex DNA could be reannealed to duplex DNA by slow cooling (30Doty P. Marmur J. Eigner J. Schildkraut C. Proc. Natl. Acad. Sci. U. S. A. 1960; 46: 461-476Crossref PubMed Google Scholar). These discoveries eventually led to the demonstration that all RNA species were formed from DNA and later contributed to the discovery of RNA splicing.During the period between 1953 and 1958, a number of reports described the existence of enzyme activities that utilized rNTPs rather than rNDPs and extended RNA chains by only a few nucleotides (specifically C and/or A residues). They were defined eventually by the tRNA terminal -CCA adding enzyme, an activity dedicated solely to the addition of C and A residues to the 3′-ends of tRNAs (31Preiss J. Dieckmann M. Berg P. J. Biol. Chem. 1961; 236: 1748-1752Abstract Full Text PDF PubMed Google Scholar, 32Furth J.J. Hurwitz J. Krug M. Alexander M. J. Biol. Chem. 1961; 236: 3317-3322Abstract Full Text PDF PubMed Google Scholar). Subsequently, it was shown that DNA sequences encoding tRNAs do not include the 3′-terminal -CCA sequence, which is essential for amino acid acylation. Uniquely, in 1960, Edmonds and Abrams reported the synthesis of poly(A) chains from ATP (33Edmonds M. Abrams R. J. Biol. Chem. 1960; 235: 1142-1149Abstract Full Text PDF PubMed Google Scholar), the first indication of poly(A) polymerase. At this time, it was evident that none of the aforementioned enzyme activities explained how RNA was synthesized. In contrast to the biosynthesis of DNA, which simplistically could be considered a single entity, the biosynthesis of RNA appeared to be more complicated because of its heterogeneity. Furthermore, there was evidence that the function of these distinct RNA species differed. It was evident that a number of specific small molecular weight RNAs (tRNAs) were carriers for individual amino acids in protein synthesis (34Hoagland M.B. Zamecnik P.C. Stephenson M.L. Biochim. Biophys. Acta. 1957; 24: 215-216Crossref PubMed Scopus (66) Google Scholar, 35Berg P. Ofengand E.J. Proc. Natl. Acad. Sci. U. S. A. 1958; 44: 78-86Crossref PubMed Google Scholar) whereas the multiple larger RNA species (ribosomal RNAs), constituting the bulk of cellular RNA, existed as a ribonucleoprotein complex where amino acids appeared to be incorporated into proteins (36Lamberg J.R. Zamecnik P.C. Biochim. Biophys. Acta. 1960; 42: 206-211Crossref PubMed Scopus (34) Google Scholar). By the late 1950s, it was generally accepted though not proven that the genetic information coded in the sequence of DNA contributed to the amino acid sequence of proteins and RNA acted as an intermediate in this process. In support of this notion, the pioneering phage work done by Seymour Cohen showed that infection of E. coli by virulent phages such as T2 caused bacterial DNA degradation that blocked bacterial protein and DNA synthesis and diverted the host to synthesis phage DNA and phage proteins (37Cohen S.S. Bacteriol. Rev. 1949; 13: 1-24Crossref PubMed Google Scholar). Similar phage experiments by Al Hershey noted that a minor RNA species was rapidly synthesized and degraded (38Hershey A.D. J. Gen. Physiol. 1953; 36: 777-789Crossref PubMed Scopus (56) Google Scholar). By far the most compelling evidence for an important role for DNA in RNA synthesis came from the experiments of Volkin and Astrachan in which T2 infection of E. coli in the presence of 32Pi resulted in the rapid formation and degradation of RNA containing the same base ratio of the T2 DNA (39Volkin E. Astrachan L. Virology. 1956; 3: 149-161Crossref Scopus (67) Google Scholar). Though I was fully aware of these results and had thought about the role of DNA in RNA synthesis in St. Louis, my efforts to screen for DNA-dependent RNA synthesis started in 1959 and were carried out with E. coli extracts. In light of my earlier confu
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