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• W2024562974 abstract "My Ph.D. thesis in the laboratory of Severo Ochoa at New York University School of Medicine in 1962 included the determination of the nucleotide compositions of codons specifying amino acids. The experiments were based on the use of random copolyribonucleotides (synthesized by polynucleotide phosphorylase) as messenger RNA in a cell-free protein-synthesizing system. At Yale University, where I joined the faculty, my co-workers and I first studied the mechanisms of protein synthesis. Thereafter, we explored the interferons (IFNs), which were discovered as antiviral defense agents but were revealed to be components of a highly complex multifunctional system. We isolated pure IFNs and characterized IFN-activated genes, the proteins they encode, and their functions. We concentrated on a cluster of IFN-activated genes, the p200 cluster, which arose by repeated gene duplications and which encodes a large family of highly multifunctional proteins. For example, the murine protein p204 can be activated in numerous tissues by distinct transcription factors. It modulates cell proliferation and the differentiation of a variety of tissues by binding to many proteins. p204 also inhibits the activities of wild-type Ras proteins and Ras oncoproteins. My Ph.D. thesis in the laboratory of Severo Ochoa at New York University School of Medicine in 1962 included the determination of the nucleotide compositions of codons specifying amino acids. The experiments were based on the use of random copolyribonucleotides (synthesized by polynucleotide phosphorylase) as messenger RNA in a cell-free protein-synthesizing system. At Yale University, where I joined the faculty, my co-workers and I first studied the mechanisms of protein synthesis. Thereafter, we explored the interferons (IFNs), which were discovered as antiviral defense agents but were revealed to be components of a highly complex multifunctional system. We isolated pure IFNs and characterized IFN-activated genes, the proteins they encode, and their functions. We concentrated on a cluster of IFN-activated genes, the p200 cluster, which arose by repeated gene duplications and which encodes a large family of highly multifunctional proteins. For example, the murine protein p204 can be activated in numerous tissues by distinct transcription factors. It modulates cell proliferation and the differentiation of a variety of tissues by binding to many proteins. p204 also inhibits the activities of wild-type Ras proteins and Ras oncoproteins. I was born in Budapest, Hungary, in 1929. As an adolescent, I wanted to study medicine, following in my grandfather's footsteps. It was he, an internist who obtained his medical training in the 1890s, who changed my mind. He was frustrated by the huge gaps in the scientific basis of medicine in the 1940s, and he advised me to study organic chemistry and biochemistry and apply what I have learned to biomedical research. Thus, I matriculated at the Technical University of Budapest and graduated in 1951 as a chemical engineer. After serving in the Hungarian army for two years, I sought admission to graduate school to study biochemistry. For this, I had to request a letter of recommendation from my professor of organic chemistry at the Technical University. Learning my career choice, the professor visibly saddened and asked, “Do you really want to spend your whole life studying proteins?” At the time, this was a reasonable question. Organic chemistry was already a well developed field, biochemistry was way behind, and molecular biology was nonexistent. That DNA was a carrier of hereditary information had been discovered only in 1944, and this discovery was still doubted by many. It was only later, in 1951, that the amino acid sequence of the first protein, the 30-amino acid-long B-chain of insulin, was determined and found to be unique by Sanger and Tuppy (1Sanger F. Tuppy H. The amino-acid sequence in the phenylalanyl chain of insulin. 2. The investigation of peptides from enzymic hydrolysates.Biochem. J. 1951; 49: 481-490Crossref PubMed Scopus (202) Google Scholar). Earlier, it had been frequently suggested that proteins may not be pure chemical entities but may consist of mixtures of closely related substances. Despite these uncertainties in the field of biochemistry at the time, I received the letter of recommendation and was accepted as a graduate student. My advisor became F. B. Straub, professor of biochemistry at the Medical University of Budapest. He was a student of Albert Szent-Györgyi, a Hungarian biochemist who was awarded the Nobel Prize for various contributions, including the discovery of vitamin C. Dr. Straub was a gifted biochemist, whose contributions included the purification and crystallization of malic and lactic dehydrogenases and the discoveries of diaphorase and the muscle protein actin (2Straub F.B. Actin.in: Szent-Györgyi A. Studies from the Institute of Medical Chemistry, University of Szeged. Volume II. S. Karger AG, Basel, Switzerland1942: 3-15Google Scholar). During the two and a half years I spent with Dr. Straub, I received my introduction to biochemistry, including a warning: before starting a research project, you must decide that you consider it worthwhile doing. One of the textbooks I used in preparation for the qualifying examination was by Fruton and Simmonds, two professors of biochemistry at Yale University. After the collapse of the Hungarian uprising against the Soviet oppression in 1956, I fled to the West, together with 200,000 Hungarians. I immigrated to the United States, which I had considered the center of biochemical research. While still in Hungary, I read with great interest about the discovery of the first enzyme capable of synthesizing RNA in the test tube: polynucleotide phosphorylase (PNP) by Grunberg-Manago and Ochoa at New York University (NYU) School of Medicine (3Grunberg-Manago M. Ochoa S. Enzymatic synthesis and breakdown of polynucleotides: polynucleotide phosphorylase.J. Am. Chem. Soc. 1955; 77: 3165-3166Crossref Google Scholar). Upon arriving in New York, I approached Dr. Ochoa. It must have been his sympathy for the cause of Hungary and my enthusiasm to become his student that induced him to accept me as a graduate student in his laboratory. Dr. Ochoa made great contributions to various fields in biochemistry, including the Krebs cycle, fatty acid synthesis, oxidative phosphorylation, propionate metabolism, and peptide chain initiation (Fig. 1) (4Ochoa S. The pursuit of a hobby.Annu. Rev. Biochem. 1980; 49: 1-30Crossref PubMed Google Scholar). He was awarded the Nobel Prize in 1959 for the discovery of PNP. He shared the prize with his former postdoctoral associate, Arthur Kornberg, who obtained it for discovering DNA polymerase. The Ochoa laboratory was a great place to learn. Lunches and coffee breaks were taken together. These were the times for discussing recent publications, results of experiments, and new ideas. My innate interest in these discussions prompted me to follow the literature diligently. The faculty member at NYU from whom I learned very much was Charlie Gilvarg. He knew everything that was worthwhile knowing. I consulted him often and missed him after his move from NYU to Princeton University. The long-standing friendship among Yoshito Kaziro, Charles Weissmann, and me started in the Ochoa laboratory. Both have had more distinguished careers than I have had. An important chapter of my research career resulted from my studies as a graduate student with Dr. Ochoa. My first assignment in his laboratory was to study the degradation of various natural RNAs by phosphorolysis catalyzed by PNP in the presence of inorganic phosphate. Thereafter, with R. W. Chambers, I established that PNP can also synthesize, from the appropriate ribonucleoside diphosphates, an RNA containing 2-thiouridylate (besides nucleotides A, C, G, and U) (5Lengyel P. Chambers R.W. Preparation of 2-thiouridine-5′-diphosphate and the enzymatic synthesis of polythiouridylic acid.J. Am. Chem. Soc. 1960; 82: 752-753Crossref Scopus (7) Google Scholar). Somewhat earlier, 2-thiouridylate was found to be present in certain natural RNAs. My next project, suggested by Dr. Ochoa, was part of his exploration of propionic acid metabolism in animal tissues. The goal was to characterize methylmalonyl isomerase from sheep kidney. This enzyme converts methylmalonyl coenzyme A, a branched chain compound, to succinyl coenzyme A, its straight chain isomer. I noted the similarity of the above conversion to that of β-methylaspartate, another branched chain compound, to glutamate, its straight chain isomer, by glutamate isomerase, as reported by Barker et al. (6Barker H.A. Weissbach H. Smyth R.D. A coenzyme containing pseudovitamin B12.Proc. Natl. Acad. Sci. U.S.A. 1958; 44: 1093-1097Crossref PubMed Google Scholar). Recognizing this similarity, I expected that methylmalonyl isomerase may require for activity the same vitamin B12 cobamide coenzymes as glutamate isomerase. In collaboration with R. Mazumder at NYU, I started to search for vitamin B12 coenzyme in our enzyme. However, prior to completing our study, three research papers reported finding vitamin B12 coenzyme in methylmalonyl isomerase. Although disappointed, we completed our study and found vitamin B12 coenzyme in our sheep kidney enzyme. We established that, of the three types of these coenzymes, only two, dimethylbenzimidazolylcobamide and benzimidazolylcobamide, appeared to fully activate the enzyme (7Lengyel P. Mazumder R. Ochoa S. Mammalian methylmalonyl isomerase and vitamin B12 coenzymes.Proc. Natl. Acad. Sci. U.S.A. 1960; 46: 1312-1318Crossref PubMed Google Scholar). Thereafter, I was supposed to join an ongoing project concerning the DNA-dependent enzymatic synthesis of RNA. However, my participation in the project was cut short. This happened because, completely unexpectedly, I became involved in the initial deciphering of the genetic code. This exciting and competitive study became the topic of my Ph.D. thesis in 1962. I recently described the detailed story of this undertaking and its origins in a prefatory chapter of the Annual Review of Microbiology. It is entitled “Memories of a senior scientist: on passing the fiftieth anniversary of the beginning of deciphering the genetic code” (8Lengyel P. Memories of a senior scientist: on passing the fiftieth anniversary of the beginning of deciphering the genetic code.Annu. Rev. Microbiol. 2012; 66: 27-38Crossref PubMed Scopus (2) Google Scholar). Thus, I will not present it here. I will just note that the two laboratories involved in the deciphering of the nucleotide compositions of codons were those of Marshall Nirenberg at the National Institutes of Health and Dr. Ochoa at NYU. The competition between the two laboratories (called by some “the code war”) accelerated the research (4Ochoa S. The pursuit of a hobby.Annu. Rev. Biochem. 1980; 49: 1-30Crossref PubMed Google Scholar, 8Lengyel P. Memories of a senior scientist: on passing the fiftieth anniversary of the beginning of deciphering the genetic code.Annu. Rev. Microbiol. 2012; 66: 27-38Crossref PubMed Scopus (2) Google Scholar, 9Nirenberg M. Historical review: deciphering the genetic code–a personal account.Trends Biochem. Sci. 2004; 29: 46-54Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). My Ph.D. thesis was entitled “Use of synthetic polynucleotides in the deciphering of the genetic code.” It included the findings in the first five articles from the series “Synthetic polynucleotides and the amino acid code.” The first article by Lengyel et al. (10Lengyel P. Speyer J.F. Ochoa S. Synthetic polynucleotides and the amino acid code.Proc. Natl. Acad. Sci. U.S.A. 1961; 47: 1936-1942Crossref PubMed Google Scholar) described the preparation of random copolynucleotides produced by PNP and containing two or more kinds of nucleotides. These were translated in a cell-free amino acid-incorporating system to reveal the nucleotide compositions of codons. The five articles reported the correct nucleotide compositions of codons for 18 amino acids. Besides the above, a short section of my thesis was based on the treatment of synthetic polyribonucleotides with nitrous acid. This revealed that the purine nucleotide I (inosinate, which contains hypoxanthine) has the same coding properties as guanylate, and the purine nucleotide X (xanthylate, which contains xanthine) is inactive in coding (11Basilio C. Wahba A.J. Lengyel P. Speyer J.F. Ochoa S. Synthetic polynucleotides and the amino acid code. V.Proc. Natl. Acad. Sci. U.S.A. 1962; 48: 613-616Crossref PubMed Google Scholar). Another short section of my thesis identified the bacterial ribosome as a target of action of the antibiotic streptomycin (12Speyer J.F. Lengyel P. Basilio C. Ribosomal localization of streptomycin sensitivity.Proc. Natl. Acad. Sci. U.S.A. 1962; 48: 684-686Crossref PubMed Google Scholar). Earlier, Erdos and Ullmann (13Erdos T. Ullmann A. Effect of streptomycin on the incorporation of amino-acids labelled with carbon-14 into ribonucleic acid and protein in a cell-free system of a mycobacterium.Nature. 1959; 183: 618-619Crossref PubMed Scopus (0) Google Scholar) reported that this antibiotic inhibited the incorporation of labeled amino acids into an extract from streptomycin-sensitive but not streptomycin-resistant bacteria. Moreover, Spotts and Stanier (14Spotts C.R. Stanier R.Y. Mechanism of streptomycin action on bacteria: a unitary hypothesis.Nature. 1961; 192: 633-637Crossref PubMed Scopus (0) Google Scholar) hypothesized that ribosomes from streptomycin-sensitive bacteria have a high affinity for streptomycin, whereas ribosomes from resistant strains have no affinity. To test their hypothesis, Speyer et al. (12Speyer J.F. Lengyel P. Basilio C. Ribosomal localization of streptomycin sensitivity.Proc. Natl. Acad. Sci. U.S.A. 1962; 48: 684-686Crossref PubMed Google Scholar) fractionated cell-free amino acid-incorporating systems from streptomycin-sensitive and streptomycin-resistant Escherichia coli into fast sedimenting ribosomal and slow sedimenting supernatant fractions. We established that when mixing one of the two types of ribosomes (streptomycin-sensitive or streptomycin-resistant) with one of the two types of supernatant fractions (streptomycin-sensitive or streptomycin-resistant), it was the ribosomal fraction (streptomycin-sensitive or streptomycin-resistant) that determined whether the mixture was sensitive or resistant to streptomycin (12Speyer J.F. Lengyel P. Basilio C. Ribosomal localization of streptomycin sensitivity.Proc. Natl. Acad. Sci. U.S.A. 1962; 48: 684-686Crossref PubMed Google Scholar). A later publication reported that several other antibiotics also impaired ribosome activity (15Vazquez D. Inhibitors of Protein Synthesis. Springer Verlag, Berlin1979Crossref Google Scholar). I ended my involvement in the studies on the genetic code at NYU in the summer of 1963, and I spent one year at the Pasteur Institute in Paris in the laboratory of the brilliant French scientist Jacques Monod (16Ullmann A. In memoriam: Jacques Monod (1910–1976).Genome Biol. Evol. 2011; 3: 1025-1033Crossref PubMed Google Scholar). At the time, the Pasteur Institute was among the prime movers of progress in molecular biology. Jaçques Monod and Francois Jacob were responsible for the discovery of regulatory genes, operators and repressors, operons, and messenger RNA and also contributed much to the understanding of allosteric regulators. Besides being a highly original and imaginative scientist, Monod was also a warm and generous man of great charm (Fig. 2) (16Ullmann A. In memoriam: Jacques Monod (1910–1976).Genome Biol. Evol. 2011; 3: 1025-1033Crossref PubMed Google Scholar). Just as in the Ochoa laboratory, lunch was eaten together in the Pasteur Institute. Scientific discussions were also just as lively, especially because the luminaries André Lwoff, Monod, and Jacob, who shared the Nobel Prize a few years later, actively participated (16Ullmann A. In memoriam: Jacques Monod (1910–1976).Genome Biol. Evol. 2011; 3: 1025-1033Crossref PubMed Google Scholar). The difference was in the source of the meals. At NYU, it was the cafeteria. This was not so in the Pasteur Institute. Although a cafeteria had just opened and was serving a variety of dishes, as well as white and red wine for lunch, the French researchers found the choice too restricted, and most of them continued to obtain their food from the neighboring charcuterie. Another difference was in the hours worked. While at NYU, I often got home after midnight. At the Pasteur Institute, at the time (perhaps to save electricity), you had to leave before 8 p.m. To stay later, you needed special permission, which was valid for only one evening. This had to be requested for me every day by Dr. Monod from the director of the Pasteur Institute, Dr. Trefouel, who personally signed it. At the Pasteur Institute, I spent several months attempting to isolate the lac repressor without success. (Three years later, in 1966, Walter Gilbert and Benno Müller-Hill accomplished the isolation (17Gilbert W. Müller-Hill B. Isolation of the lac repressor.Proc. Natl. Acad. Sci. U.S.A. 1966; 56: 1891-1898Crossref PubMed Google Scholar).) My subsequent efforts in Paris were devoted to the development of an experimental system for a new project I wanted to start after my return to NYU. After having studied the genetic code (i.e. the dictionary used in the translation of mRNA into protein), I wished to explore the mechanisms of translation, especially that of peptide chain elongation. Three elongation factors had just been identified by Lucas-Lenard and Lipmann (18Lucas-Lenard J. Lipmann F. Separation of three microbial amino acid polymerization factors.Proc. Natl. Acad. Sci. U.S.A. 1966; 55: 1562-1566Crossref PubMed Google Scholar). The obstacle toward further progress seemed to be that one of the three (designated as elongation factor EF-Ts) appeared to be unstable after purification. I hoped that the thermophilic microorganism Bacillus stearothermophilus, growing at temperatures up to 70 °C, would be a source of factors stable for purification at lower temperatures (19Arca M. Calvori C. Frontali L. Tecce G. The enzymic synthesis of aminoacyl derivatives of soluble ribonucleic acid from Bacillus stearothermophilus.Biochim. Biophys. Acta. 1964; 87: 440-448PubMed Google Scholar). B. stearothermophilus turned out to be a good choice. With gloves to prevent burning one's fingers, B. stearothermophilus was convenient to work with: it had a generation time of <15 min, and at a temperature of growth of 70 °C, there was no need for sterility. After a year in Paris, I returned to NYU as an assistant professor. At NYU, Israel Algranati, a visiting scientist from Argentina, joined me to continue the experiments with B. stearothermophilus. The thermal stability and high activity at 65 °C of the B. stearothermophilus system, as well as its low nuclease activity, allowed the translation at 65 °C of polynucleotides (e.g. 1.4:1 copolymer of adenylic and uridylic acids), which, because of their highly ordered secondary and tertiary structures, are almost inactive at 37 °C in an E. coli system (20Algranati I.D. Lengyel P. Polynucleotide-dependent incorporation of amino acids in a cell-free system from thermophilic bacteria.J. Biol. Chem. 1966; 241: 1778-1783Abstract Full Text PDF PubMed Google Scholar). In the fall of 1965, I joined the faculty of the Molecular Biophysics and Biochemistry Department at Yale. I believe that my invitation to the department by its chair, Fred Richards, might have originated from a series of conversations I had with Alan Garen from the department, among others, at several Cold Spring Harbor meetings and a meeting in Hyderabad, India. These conversations concerned the genetic code, a topic of interest for both of us. My collaborators Art Skoultchi, Yasushi Ono, and Hong Mo Moon isolated, from the thermophilic B. stearothermophilus, three elongation factors designated in the now accepted way: EF-Ts, EF-G, and EF-Tu (at the time, we designated them as S1, S2, and S3, respectively). All three factors were needed for the poly(U)-promoted formation of polyphenylalanine from Phe-tRNA (21Skoultchi A. Ono Y. Moon H.M. Lengyel P. On three complementary amino acid polymerization factors from Bacillus stearothermophilus: separation of a complex containing two of the factors, guanosine-5′-triphosphate and aminoacyl-transfer RNA.Proc. Natl. Acad. Sci. U.S.A. 1968; 60: 675-682Crossref PubMed Google Scholar). To make our studies more similar to those of Lucas-Lenard and Lipmann (18Lucas-Lenard J. Lipmann F. Separation of three microbial amino acid polymerization factors.Proc. Natl. Acad. Sci. U.S.A. 1966; 55: 1562-1566Crossref PubMed Google Scholar), we formed a ribosome·poly(U)·acetyl-Phe-tRNA complex in vitro. The acetyl-Phe-tRNA served as an analog of the natural chain initiator fMet-tRNAf. There were indications that an acetyl-Phe-tRNA·GTP·EF-Tu complex (ternary complex) can carry, in the presence of poly(U), Phe-tRNA to the ribosome. Ono et al. (22Ono Y. Skoultchi A. Waterson J. Lengyel P. Peptide chain elongation: GTP cleavage catalysed by factors binding aminoacyl-transfer RNA to the ribosome.Nature. 1969; 222: 645-648Crossref PubMed Scopus (0) Google Scholar, 23Ono Y. Skoultchi A. Waterson J. Lengyel P. Stoichiometry of aminoacyl-transfer RNA binding and GTP cleavage during chain elongation and translocation.Nature. 1969; 223: 697-701Crossref PubMed Scopus (0) Google Scholar) isolated such a complex and established that the molar ratio of GTP to Phe-tRNA was 1:1 and that EF-Tu can form a ternary complex with each aminoacyl-tRNA involved in chain elongation. It was known that the chain initiator in E. coli is fMet-tRNAf synthesized by formylation of Met-tRNAf. A different Met-tRNA (Met-tRNAm) provides Met for internal positions of the peptide chain (24Adams J.M. Capecchi M.R. N-Formylmethionyl-sRNA as the initiator of protein synthesis.Proc. Natl. Acad. Sci. U.S.A. 1966; 55: 147-155Crossref PubMed Google Scholar). It was also known that as initiator codons in E. coli, GUG and AUG specify fMet-tRNAf; as codons for internal amino acid residues, GUG stands for Val-tRNA, and AUG stands for Met-tRNAm (25Clark B.F. Marcker K.A. The role of N-formyl-methionyl-sRNA in protein biosynthesis.J. Mol. Biol. 1966; 17: 394-406Crossref PubMed Google Scholar). The dual specificity of GUG raised the question of how mix-ups between Val-tRNA, fMet-tRNAf, and Met-tRNAf are avoided during chain elongation. As noted, aminoacyl-tRNA·GTP·EF-Tu complexes are intermediates in binding aminoacyl-tRNA to the ribosomes. Ono et al. (26Ono Y. Skoultchi A. Klein A. Lengyel P. Peptide chain elongation: discrimination against the initiator transfer RNA by microbial amino-acid polymerization factors.Nature. 1968; 220: 1304-1307Crossref PubMed Scopus (0) Google Scholar) established that EF-Tu and GTP do not form a complex with either Met-tRNAf or fMet-tRNAf. This explains why Met residues are not inserted into the polypeptide chain in response to internal GUG codons. We examined the fate of the three components of the ternary complex after the transfer of its Phe-tRNA moiety to the ribosome·poly(U)·acetyl-Phe-tRNA complex. We established that one GTP molecule was cleaved for each Phe-tRNA bound to the ribosome, and this cleavage was promoted by EF-Tu in the complex. Cleavage of an additional GTP is needed for translocation (23Ono Y. Skoultchi A. Waterson J. Lengyel P. Stoichiometry of aminoacyl-transfer RNA binding and GTP cleavage during chain elongation and translocation.Nature. 1969; 223: 697-701Crossref PubMed Scopus (0) Google Scholar). The GTP cleavage product Pi was released in a free state, but the other, GDP, was released in complex with EF-Tu. Skoultchi et al. (27Skoultchi A. Ono Y. Waterson J. Lengyel P. Peptide chain elongation.Cold Spring Harbor Symp. Quant. Biol. 1969; 34: 437-454Crossref PubMed Google Scholar, 28Skoultchi A. Ono Y. Waterson J. Lengyel P. Peptide chain elongation; indications for the binding of an amino acid polymerization factor, guanosine 5′-triphosphate–aminoacyl transfer ribonucleic acid complex to the messenger-ribosome complex.Biochemistry. 1970; 9: 508-514Crossref PubMed Google Scholar) tested if GTP cleavage is a prerequisite for peptide bond formation in an experiment in which we substituted for GTP an analog, guanosine 5′-(β,γ-methylenetriphosphate) (GMPPCP), which has a methylene bridge instead of oxygen between the β- and γ-phosphorus atoms, so it cannot be cleaved enzymatically into GDP and Pi. In this case, equimolar amounts of EF-Tu, Phe-tRNA, and GMPPCP were bound to the ribosome, but no dipeptide was formed. GTP cleavage is required for and has to precede the release of EF-Tu from the ribosome and peptide bond formation (27Skoultchi A. Ono Y. Waterson J. Lengyel P. Peptide chain elongation.Cold Spring Harbor Symp. Quant. Biol. 1969; 34: 437-454Crossref PubMed Google Scholar, 28Skoultchi A. Ono Y. Waterson J. Lengyel P. Peptide chain elongation; indications for the binding of an amino acid polymerization factor, guanosine 5′-triphosphate–aminoacyl transfer ribonucleic acid complex to the messenger-ribosome complex.Biochemistry. 1970; 9: 508-514Crossref PubMed Google Scholar). In 1966, Eisenstadt and I proved that fMet-tRNAf is required for the translation of bacteriophage f2 RNA in an E. coli extract (29Eisenstadt J. Lengyel P. Formylmethionyl-tRNA dependence of amino acid incorporation in extracts of trimethoprim-treated Escherichia coli.Science. 1966; 154: 524-527PubMed Google Scholar). The chain initiator codons AUG and GUG also specify internal amino acid residues. The characteristics that determine whether an AUG or GUG codon in an mRNA serves as a signal for initiating a new peptide chain were not known. Kondo et al. (30Kondo M. Eggerston G. Eisenstadt J. Lengyel P. Ribosome formation from subunits: dependence on formylmethionyl-transfer RNA in extracts from E. coli.Nature. 1968; 220: 368-371Crossref PubMed Scopus (0) Google Scholar) developed a procedure for isolating a chain initiation signal containing segments of mRNA. It was known that the region of mRNA that is bound to ribosomes is protected against cleavage by nucleases. Furthermore, the intermediates in the assembly of a peptide chain initiation complex had been reported. First, a 30 S ribosomal subunit forms a complex with mRNA in the presence of GTP and initiation factors (31Nomura M. Lowry C.V. Phage f2 RNA-directed binding of formylmethionyl-tRNA to ribosomes and the role of 30 S ribosomal subunits in initiation of protein synthesis.Proc. Natl. Acad. Sci. U.S.A. 1967; 58: 946-953Crossref PubMed Google Scholar). Subsequently, the 50 S ribosomal subunit is attached, forming a 70 S initiation complex. We expected that the binding of mRNA in a 70 S initiation complex is dependent on fMet-tRNAf, and if fMet-tRNAf is the only aminoacyl-tRNA present, the ribosome has to bind uniquely to the initiation signal (30Kondo M. Eggerston G. Eisenstadt J. Lengyel P. Ribosome formation from subunits: dependence on formylmethionyl-transfer RNA in extracts from E. coli.Nature. 1968; 220: 368-371Crossref PubMed Scopus (0) Google Scholar). We developed conditions in which bacteriophage f2 RNA did not become bound in a 70 S complex in the absence of fMet-tRNAf. Moreover, in the presence of fMet-tRNAf, only a single ribosome was attached to the f2 RNA molecule. Gupta et al. (32Gupta S.L. Chen J. Schaefer L. Weissman S.M. Lengyel P. Studies on the nucleotide sequence of a ribosome attachment site of bacteriophage f2 RNA.Cold Spring Harbor Symp. Quant. Biol. 1969; 34: 630-633PubMed Google Scholar) isolated such a 70 S complex containing f2 RNA, treated it with ribonuclease, and recovered the 61-nucleotide-long segment of the f2 RNA that was protected against nuclease action by the attached ribosome. We presented only a partial sequence of this segment at a Cold Spring Harbor meeting. At the same meeting, Joan Steitz, who also used the approach of Kondo et al. (30Kondo M. Eggerston G. Eisenstadt J. Lengyel P. Ribosome formation from subunits: dependence on formylmethionyl-transfer RNA in extracts from E. coli.Nature. 1968; 220: 368-371Crossref PubMed Scopus (0) Google Scholar) to obtain the initiation signal containing the segment of bacteriophage R17 RNA, presented its complete sequence, including the initiation signal (33Steitz J.A. Nucleotide sequences of the ribosomal binding sites of bacteriophage R17 RNA.Cold Spring Harbor Symp. Quant. Biol. 1969; 34: 621-630Crossref PubMed Google Scholar). We presented the complete sequence of the initiation signal containing the segment of f2 RNA subsequently (34Gupta S.L. Chen J. Schaefer L. Lengyel P. Weissman S.M. Nucleotide sequence of a ribosome attachment site of bacteriophage f2 RNA.Biochem. Biophys. Res. Commun. 1970; 39: 883-888Crossref PubMed Google Scholar). Having established the conditions for attaching a ribosome to a single initiation signal on an mRNA enabled us to study the movement of the ribosome during peptide chain elongation. Gupta et al. (35Gupta S.L. Waterson J. Sopori M.L. Weissman S.M. Lengyel P. Movement of the ribosome along the messenger ribonucleic acid during protein synthesis.Biochemistry. 1971; 10: 4410-4421Crossref PubMed Google Scholar) determined that (a) one step of the ribosome along the mRNA is (as expected) 3 nucleotides long, and (b) the movement of the ribosome along the mRNA during peptide chain elongation is triggered by EF-G and GTP. The experiments on which these conclusions were based included the following. A single ribosome was bound to bacteriophage f2 RNA in the presence of fMet-tRNAf under conditions in which it is bound to the coat protein initiation site (initiation complex (IC)). An aliquot of the IC was c" @default.
• W2024562974 created "2016-06-24" @default.
• W2024562974 creator A5056435911 @default.
• W2024562974 date "2014-07-01" @default.
• W2024562974 modified "2023-10-03" @default.
• W2024562974 title "Wanderings in Biochemistry" @default.
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