From Polynucleotide Phosphorylase to Neurobiology
2005; Elsevier BV; Volume: 280; Issue: 47 Linguagem: Inglês
10.1074/jbc.x500007200
ISSN1083-351X
Autores Tópico(s)Mitochondrial Function and Pathology
ResumoIn the fall of 1944, I enrolled at the Hebrew University of Jerusalem. The student body numbered about 700 and the choice of faculties was somewhat limited. My hope was to study medicine, but the plans to open a medical school were still at the drawing board stage. I therefore chose to study chemistry with biochemistry and bacteriology as minor subjects, which I thought I would need later if I went into medicine. As employment opportunities were limited in British Mandatory Palestine, it was hoped that university training would help me get a job in the food technology industry or at the Dead Sea potash industry. When I started as a student, the structure and function of DNA and RNA were not known. Thymonucleic acid (DNA) had been isolated from thymus and pus, and zymonucleic acid (RNA) was found in yeast. The tetranucleotide hypothesis for DNA suggested by Phoebus Aaron Theodor Levene still prevailed, and there were speculations about branched chain as distinct from linear structures for RNA. Nucleic acids were also considered as nothing more than a storehouse of phosphorus for the varying requirements of the cell. However, the role of DNA in bacterial transformation, demonstrated by Oswald Theodore Avery, Colin MacLeod, and Maclyn McCarty in 1944, was discussed extensively during our bacteriology courses. Within a few years, I had the good fortune to enter the nucleic acid field where progress had evolved dramatically. In June 1949, upon the completion of my studies at the Hebrew University of Jerusalem, Professor Ernst David Bergmann, the Scientific Director of the Weizmann Institute of Science, invited me to join him as his Ph.D. student. I gladly accepted and moved to Rehovot, which was then a small village with about 7000 inhabitants. Ernst Bergmann was an organic chemist, a former student of the noted German chemist, Wilhelm Schlenk, and co-worker of Professor Chaim Weizmann, the first President of the State of Israel. Bergmann had a dynamic and brilliant personality with an encyclopedic knowledge of chemistry and a broad interest in science (1Ginsburg D. Ernst David Bergmann, an appreciation on the occasion of his sixtieth birthday..Isr. J. Chem. 1963; 1: 323-350Crossref Scopus (7) Google Scholar). For my doctoral dissertation, Bergmann recommended I choose a subject close to Chaim Weizmann's scientific interests, namely the mechanism of pentose fermentation in bacteria. While I was in the advanced stages of my doctoral work, Sol Spiegelman from the Department of Microbiology of the University of Illinois, Urbana came to Rehovot to consider an offer that had been made to him to join the Weizmann Institute. Spiegelman suggested that for my postdoctoral studies I contact Arthur Kornberg, Head of the Department of Microbiology of the Washington University School of Medicine in St. Louis, Missouri. Kornberg had just moved from the National Institutes of Health (NIH) and was recruiting people for his new department. I was greatly impressed by Arthur Kornberg's early publications on coenzyme and nucleotide synthesis and decided to write and ask if he would accept me as a postdoctoral fellow. Spiegelman offered to talk to Kornberg on my behalf, and on the strength of his recommendation, I had the good fortune to be accepted and to receive a fellowship from the Dazian Foundation. In March 1955, I arrived in St. Louis to join Arthur Kornberg's laboratory. He proposed that I try to construct a cell-free system that would catalyze the synthesis of RNA. As substrate, I used 14C-labeled ATP, which I had to synthesize myself from 14C-labeled adenine by a series of enzymatic reactions because commercially labeled nucleotides were not available then. Within a short time, I was able to construct a cell-free system from Escherichia coli cells that converted 14C-labeled ATP to an acid-insoluble polyribonucleotide. Moreover, the addition of adenylate kinase (myokinase) to the system increased the rate of the reaction. Although the activity was barely detectable, Kornberg thought that I should attempt to characterize the polynucleotide-synthesizing system. While making rapid progress in purifying the E. coli enzyme, we learned from Herman Kalckar, an eminent Danish biochemist who came for a visit, that Marianne Grunberg-Manago and Severo Ochoa at New York University had independently discovered an activity, similar to ours, in extracts of Azotobacter vinelandii (A. agilis). The enzyme was named polynucleotide phosphorylase (PNPase) and was shown to convert nucleoside diphosphates into polynucleotides. Although we were disappointed by the news of their findings, we decided to continue our studies with the purified E. coli enzyme. Acting on this new information, we shifted to using ADP rather than ATP and found it to be the preferred substrate in our system (2Grunberg-Manago M. Ortiz P.J. Ochoa S. Enzymic synthesis of polynucleotides. I. Polynucleotide phosphorylase of Azotobacter vinelandii..Biochim. Biophys. Acta. 1956; 20: 269-285Crossref PubMed Scopus (70) Google Scholar, 3Littauer U.Z. Kornberg A. Reversible synthesis of polyribonucleotides with an enzyme from Escherichia coli..J. Biol. Chem. 1957; 226: 1077-1092Abstract Full Text PDF PubMed Google Scholar). PNPase was the first enzyme to be discovered that catalyzes the de novo synthesis of polyribonucleotides with a 3′,5′-phosphodiester bond, and its discovery stimulated a considerable number of investigations. The cumulative studies have established that in the forward reaction long polyribonucleotides (pN)n are synthesized in a processive fashion from various ribonucleoside diphosphates (ppN) with concomitant release of inorganic phosphate (Pi). Each of the four common ribonucleoside diphosphates can serve separately as a substrate for the polymerization reaction, leading to formation of homopolymers. Polymerization of a mixture of ribonucleoside diphosphates that contains different bases results in a random copolymer. The enzyme does not require a template and cannot copy one. In the reverse reaction, PNPase is a processive 3′ to 5′ exoribonuclease that catalyzes the stepwise phosphorolysis of single-stranded polyribonucleotides, liberating ribonucleoside diphosphates. n·ppN⇄(pN)n+n·Pi In the processive phosphorolysis of long chain polyribonucleotides, the enzyme tends to degrade a single chain to completion, releasing ribonucleoside diphosphates plus a resistant short oligoribonucleotide before commencing phosphorolysis of another chain. In contrast, short oligoribonucleotides are degraded by a random nonprocessive mechanism in which the enzyme dissociates from the substrate after the phosphorolysis of each nucleotide (reviewed in Refs. 4Littauer U.Z. Soreq H. Polynucleotide phosphorylase..in: Boyer P.D. The Enzyme. Vol. 15. Academic Press, New York1982: 517-553Google Scholar and 5Littauer U.Z. Grunberg-Manago M. Polynucleotide phosphorylase..in: Creighton T.E. Encyclopedia of Molecular Biology. Vol. 3. John Wiley and Sons, New York1999: 1911-1918Google Scholar). PNPase was also found to catalyze an exchange reaction between free in organic phosphate and the β-phosphate of several ribonucleoside diphosphates. This reaction is apparently a result of combined polymerization and phosphorolytic reactions that occur under equilibrium conditions (2Grunberg-Manago M. Ortiz P.J. Ochoa S. Enzymic synthesis of polynucleotides. I. Polynucleotide phosphorylase of Azotobacter vinelandii..Biochim. Biophys. Acta. 1956; 20: 269-285Crossref PubMed Scopus (70) Google Scholar, 3Littauer U.Z. Kornberg A. Reversible synthesis of polyribonucleotides with an enzyme from Escherichia coli..J. Biol. Chem. 1957; 226: 1077-1092Abstract Full Text PDF PubMed Google Scholar, 4Littauer U.Z. Soreq H. Polynucleotide phosphorylase..in: Boyer P.D. The Enzyme. Vol. 15. Academic Press, New York1982: 517-553Google Scholar, 6Kimhi Y. Littauer U.Z. Purification and properties of polynucleotide phosphorylase from Escherichia coli..J. Biol. Chem. 1968; 243: 231-240Abstract Full Text PDF PubMed Google Scholar). Under suitable conditions, the enzyme also catalyzes the elongation of a primer oligoribonucleotide with a free 3′-terminal hydroxyl group (7Leder P. Singer M.F. Brimacombe R.L. Synthesis of trinucleoside diphosphates with polynucleotide phosphorylase..Biochemistry. 1965; 4: 1561-1567Crossref PubMed Scopus (35) Google Scholar, 8Moses R.E. Singer M.F. Polynucleotide phosphorylase of Micrococcus luteus. Studies on the polymerization reaction catalyzed by primer-dependent and primer-independent enzymes..J. Biol. Chem. 1970; 245: 2414-2422Abstract Full Text PDF PubMed Google Scholar). At that early stage, we pointed out that it is not apparent how an enzyme that appears to polymerize the available ribonucleoside diphosphates in a random fashion produces the specific nucleotide composition of the RNA of a given species (3Littauer U.Z. Kornberg A. Reversible synthesis of polyribonucleotides with an enzyme from Escherichia coli..J. Biol. Chem. 1957; 226: 1077-1092Abstract Full Text PDF PubMed Google Scholar). Subsequent research in other laboratories showed that the cellular function of the enzyme is to degrade RNA and that RNA synthesis is catalyzed by DNA-directed RNA polymerase. In the spring of 1956, the enzyme was already purified about 300-fold when Arthur informed me that Leon Heppel, then Head of the Section for Metabolic and Arthritis Diseases at the NIH, was interested in the enzyme. Leon then came for a visit of a few days to get acquainted with our purification procedure and assay systems. Leon, devoted to his scientific work, did not waste a minute and spent long hours into the night working in our laboratory. Prior to his return to Bethesda, I gave him some of our purified PNPase and an "activator" fraction. Toward the end of my stay in St.Louis, Alex Rich and Leon Heppel invited me to the NIH to acquaint them with my PNPase purification procedure and the synthesis of long polynucleotides. While there I also examined the phosphorolysis of several RNA preparations, and together with Gary Felsenfeld and Alex Rich we analyzed the various synthetic polyribonucleotides in the ultracentrifuge. The sedimentation data indicated molecular weights of about 400,000. They later obtained some excellent x-ray diffraction pictures from the polynucleotides. Heppel was very generous in providing me with samples of his RNA and enzyme collection, which I intended to use upon my return to the Weizmann Institute. In our experiments PNPase was found to readily catalyze the phosphorolysis of synthetic polyribonucleotides as well as high molecular weight RNA preparations, whereas commercial and low molecular weight RNA samples were not attacked to a significant extent (3Littauer U.Z. Kornberg A. Reversible synthesis of polyribonucleotides with an enzyme from Escherichia coli..J. Biol. Chem. 1957; 226: 1077-1092Abstract Full Text PDF PubMed Google Scholar). I suspected that the RNA samples were degraded products that were resistant to phosphorolysis by the enzyme. Later, in Rehovot, these observations led me to develop a method for isolating intact bacterial and mammalian RNA. Analysis in the ultracentrifuge of the E. coli preparations revealed the existence of several types of RNA with sedimentation constants of 4.1, 16.5, and 23.7S. The two high molecular weight RNA components were separated from the low molecular weight fraction by ammonium sulfate precipitation and turned out to be derived from ribosomes. These were not trivial findings because a number of investigators considered ribosomal RNA (rRNA) as an aggregate of short polynucleotides and did not realize the inherent instability of RNA compared with DNA. As expected the high molecular weight rRNA components were efficiently phosphorolyzed by PNPase. On the other hand, transfer RNA (tRNA) preparations were attacked more slowly and to a limited extent (20-30%). Urea was found to increase the degree of breakdown of tRNA, indicating that the secondary structure of tRNA hinders the enzymatic attack (9Littauer U.Z. Eisenberg H. Ribonucleic acid from Escherichia coli preparation, characterization and physical properties..Biochim. Biophys. Acta. 1959; 32: 320-337Crossref PubMed Scopus (36) Google Scholar, 10Littauer U.Z. The unfolding of our understanding of RNA structure: a personal reflection..Biophys. Chem. 2000; 86: 259-266Crossref PubMed Scopus (5) Google Scholar). Having at hand intact rRNA preparations, I suggested to my good friend and colleague, Heini Eisenberg, that we collaborate in an attempt to determine their physical properties. At that time the structure of RNA, unlike that of double-stranded DNA, was unknown mainly because of the lack of undegraded RNA preparations and because the existence of several types of RNA was not yet realized. Early attempts to secure clear diffraction patterns of RNA had failed (11Rich A. Watson J.D. Physical studies on ribonucleic acid..Nature. 1954; 173: 995-996Crossref PubMed Scopus (16) Google Scholar). We soon discovered the single-stranded nature of rRNA and the ways in which it differs from double-stranded DNA. Thus, viscosity and birefringence of flow measurements showed that rRNA is quite sensitive to increasing the ionic strength of the solution in contrast with double-stranded DNA, which shows a much smaller dependence on ionic strength concentrations. We proposed, therefore, that rRNA behaves as a flexible, contractile single-stranded coil and that each ribosomal subunit contains a single continuous uninterrupted RNA chain (reviewed in Ref. 10Littauer U.Z. The unfolding of our understanding of RNA structure: a personal reflection..Biophys. Chem. 2000; 86: 259-266Crossref PubMed Scopus (5) Google Scholar). Further experiments on rRNA were performed in collaboration with Robert Cox (a visiting scientist now at the National Institute for Medical Research, Mill Hill). Our studies showed a close correlation of the ionic strength dependence of optical rotation, optical density, and hydrodynamic properties. These early results indicated that rRNA possessed a significant secondary structure, a rather novel observation for its time. Several years later we demonstrated together with my graduate student, Inder M. Verma (now a Professor and leading molecular biologist at the Salk Institute), and Marvin Edelman (then a visiting scientist from Harvard Medical School) the presence of rRNA in mitochondria from several fungal species. We also showed that the mitochondrial rRNA possesses a unique ordered structure that differs from that of the homologous cytoplasmic rRNA (reviewed in Refs. 10Littauer U.Z. The unfolding of our understanding of RNA structure: a personal reflection..Biophys. Chem. 2000; 86: 259-266Crossref PubMed Scopus (5) Google Scholar and 12Littauer U.Z. RNA enzymology and beyond..in: Semenza G. Turner A.J. Comprehensive Biochemistry. Vol. 42. Elsevier Science Publishers B.V., Amsterdam2003: 221-284Google Scholar). By this time, we had become interested in understanding the mechanisms that govern the regulation of tRNA and mRNA activity. We devised methods for the purification of specific tRNA species and studied the post-transcriptional modification of tRNA chains. In particular we were intrigued by the high content of modified bases in tRNA. We showed that methylated bases in tRNA are not likely to be essential for cell viability but depending on the type of base modification and position along the tRNA chain they may play a role in the fine tuning of tRNA activity (12Littauer U.Z. RNA enzymology and beyond..in: Semenza G. Turner A.J. Comprehensive Biochemistry. Vol. 42. Elsevier Science Publishers B.V., Amsterdam2003: 221-284Google Scholar). The late Violet Daniel, my first graduate student (who later became a Weizmann Institute Professor of Biochemistry), joined my laboratory in 1957. She purified and characterized tRNA nucleotidyltransferase from rat liver. The enzyme was found to have an important role in the proofreading and repair of the universal 3′-CCA end of tRNA. Further experiments by the late Jacov Tal (until recently Professor and Head of the Virology Department at Ben-Gurion University) showed that the enzyme adds CMP to tRNA... N by a nonprocessive mechanism. Moreover, together with Violet and colleagues we developed a novel method that allowed monitoring the hybridization of individually labeled aminoacylated tRNA species with DNA. Using that method, we were able to reveal the presence of several unique T4 phage-coded tRNA species (13Daniel V. Sarid S. Littauer U.Z. Bacteriophage induced transfer RNA in Escherichia coli. New transfer RNA molecules are synthesized on the bacteriophage genome..Science. 1970; 167: 1682-1688Crossref PubMed Scopus (58) Google Scholar). Our discovery was well received during an EMBO workshop on tRNA that was organized by Sydney Brenner in Cambridge, UK, in March 1969. In another project that involved Violet Daniel, Jacques S. Beckmann, Sara Sarid, Jacob I. Grimberg, and Max Herzberg we were the first to isolate a tRNA gene (14Daniel V. Beckmann J.S. Sarid S. Grimberg J.I. Herzberg M. Littauer U.Z. Purification and in vitro transcription of a transfer RNA gene..Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2268-2272Crossref PubMed Scopus (12) Google Scholar). Simultaneously, our studies with PNPase have continued. Together with Yosef Kimhi, another graduate student (who became later Vice President of Scientific Affairs, Yeda Co.), we purified further the E. coli enzyme. Evidence was obtained to support our hypothesis that the same enzyme catalyzes the nucleotide polymerization and the ADP-Pi exchange reaction (6Kimhi Y. Littauer U.Z. Purification and properties of polynucleotide phosphorylase from Escherichia coli..J. Biol. Chem. 1968; 243: 231-240Abstract Full Text PDF PubMed Google Scholar). In addition, the intracellular distribution of PNPase was examined in E. coli cells. We showed that the major part of the enzyme activity (80%) is present in the soluble fraction; 10% of the total activity is bound to the cell membrane; and about 10% remains bound to washed ribosomes (15Kimhi Y. Littauer U.Z. The intracellular distribution of polynucleotide phosphorylase in Escherichia coli cells..Biochemistry. 1967; 6: 2066-2073Crossref PubMed Scopus (6) Google Scholar). Several years later Hermona (Mona) Soreq, a graduate student (now Professor and Head of the Institute of Life Sciences at the Hebrew University of Jerusalem), succeeded in purifying E. coli PNPase to homogeneity (16Soreq H. Littauer U.Z. Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3′ sequences of RNA..J. Biol. Chem. 1977; 252: 6885-6888Abstract Full Text PDF PubMed Google Scholar). The purified enzyme was virtually free of contaminating nucleases, which allowed us to use its 3′-exonucleolytic activity to determine the size and composition of the 3′-terminal sequences of RNA molecules and their function. Thus, with a molar excess of PNPase over the substrate a synchronous mode of phosphorolysis is established in which NDP molecules are sequentially released from the 3′ terminus of the RNA chains (4Littauer U.Z. Soreq H. Polynucleotide phosphorylase..in: Boyer P.D. The Enzyme. Vol. 15. Academic Press, New York1982: 517-553Google Scholar, 17Soreq H. Nudel U. Salomon R. Revel M. Littauer U.Z. In vitro translation of polyadenylic acid-free rabbit globin messenger RNA..J. Mol. Biol. 1974; 88: 233-245Crossref PubMed Scopus (74) Google Scholar). Moreover, we observed that at 0 °C, the poly(A) tails of mRNA molecules are readily phosphorolyzed, whereas the deadenylated mRNA chains remain intact. Together with Uri Nudel, Raphael Salomon, and Michel Revel we found that globin poly(A)-free mRNA could still be translated in a Krebs ascites tumor cell-free extract (the nuclease-treated reticulocyte lysate cell-free system had not yet been developed). We also observed that at long periods of incubation, the rate of globin synthesis appeared to level off more sharply with deadenylated mRNA than with native mRNA (17Soreq H. Nudel U. Salomon R. Revel M. Littauer U.Z. In vitro translation of polyadenylic acid-free rabbit globin messenger RNA..J. Mol. Biol. 1974; 88: 233-245Crossref PubMed Scopus (74) Google Scholar). The in vitro systems survive no longer than 2 h and are inadequate for detection of long term effects of the poly(A) tail on mRNA stability. We, therefore, turned our attention to the use of Xenopus laevis oocytes and were fortunate to collaborate with Georges Huez and Gérard Marbaix from the Free University of Brussels, who were experts in the use of this relatively new system. To examine the functional stability (i.e. ability to be translated) of the mRNA, deadenylated mRNA samples were microinjected into the oocytes. The results showed that the rate of globin synthesis with poly(A)-free mRNA is considerably lower than with native mRNA, and this difference became more pronounced at longer periods of incubation (18Huez G. Marbaix G. Hubert E. Leclercq M. Nudel U. Soreq H. Salomom R. Lebleu B. Revel M. Littauer U.Z. Role of the polyadenylate segment in the translation of globin messenger RNA in Xenopus oocytes..Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3143-3146Crossref PubMed Scopus (118) Google Scholar). In subsequent experiments we were able to show that readenylation of poly(A)-free globin mRNA restores its functional stability. In retrospect, we were more than fortunate in choosing globin mRNA for our studies, as there are examples where the removal of poly(A) tracts from some other mRNA species does not affect their stability (reviewed in Ref. 19Littauer U.Z. Soreq H. The regulatory function of poly(A) and adjacent 3′ sequences in translated RNA..Prog. Nucleic Acids Res. Mol. Biol. 1982; 27: 53-83Crossref PubMed Scopus (58) Google Scholar). Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by Uri Nudel and Hermona Soreq. This was accomplished by partial phosphorolysis of mRNA with the purified E. coli PNPase. By varying the salt concentration and the time of incubation of the phosphorolysis reaction mixture, as well as performing oligo(dT)-cellulose chromatography at different temperatures, globin mRNA preparations with poly(A) tails of varying size were obtained. In collaboration with our Belgian colleagues, Gérard Marbaix, Georges Huez, Madeleine Leclercq, Evelyne Hubert, and Hubert Chantrenne, the functional stability of these molecules was examined in Xenopus oocytes. Globin mRNA molecules with a segment of 32 or more adenylate residues had equivalent functional stability, whereas those with less than 32 adenylate residues were 10-fold less stable. We suggested that a minimal size of the poly(A) segment is essential for attaching to poly(A)-binding proteins (PABP), thereby protecting the mRNA from nucleolytic degradation (20Nudel U. Soreq H. Littauer U.Z. Marbaix G. Huez G. Leclercq M. Hubert E. Chantrenne H. Globin mRNA species containing poly(A) segments of different lengths. Their functional stability in Xenopus oocytes..Eur. J. Biochem. 1976; 64: 115-121Crossref PubMed Scopus (130) Google Scholar). This suggestion correlates well with the observation of Bradford Baer and Roger Kornberg that the minimal length of the poly(A) tail necessary for PABP binding is 27 residues (21Baer B.W. Kornberg R.D. The protein responsible for the repeating structure of cytoplasmic poly(A)-ribonucleoprotein..J. Cell Biol. 1983; 96: 717-721Crossref PubMed Scopus (118) Google Scholar). To account for the great variability among mRNA species, it was proposed that the 3′-untranslated region (3′-UTR) can modulate the affinity of PABP for the poly(A) segment, thus permitting control of the poly(A) stability in individual mRNA species (22Littauer U.Z. Co-chairman's remarks: reflections on RNA..Gene (Amst.). 1993; 135: 209-214Crossref PubMed Scopus (4) Google Scholar). It is also apparent that the interaction between the poly(A) tail-PABP complex and cap-associated translation initiation factors may be important in maintaining the physical integrity of mRNA (23Deo R.C. Bonanno J.B. Sonenberg N. Burley S.K. Recognition of polyadenylate RNA by the poly(A)-binding protein..Cell. 1999; 98: 835-845Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). Thus, there is a multitude of systems that use the poly(A) tract to control the expressions of specific mRNA species. The number of cis-acting elements and trans-acting factors regulating turnover of mRNA is increasing rapidly, and the complexity of these processes grows in parallel. We also used the 3′-exonucleolytic activity of PNPase to determine the size of the poly(A) tails from various mRNA species (17Soreq H. Nudel U. Salomon R. Revel M. Littauer U.Z. In vitro translation of polyadenylic acid-free rabbit globin messenger RNA..J. Mol. Biol. 1974; 88: 233-245Crossref PubMed Scopus (74) Google Scholar, 20Nudel U. Soreq H. Littauer U.Z. Marbaix G. Huez G. Leclercq M. Hubert E. Chantrenne H. Globin mRNA species containing poly(A) segments of different lengths. Their functional stability in Xenopus oocytes..Eur. J. Biochem. 1976; 64: 115-121Crossref PubMed Scopus (130) Google Scholar, 24Grosfeld H. Soreq H. Littauer U.Z. Membrane associated cytoplasmic mRNA in Artemia salina; functional and physical changes during development..Nucleic Acids Res. 1977; 4: 2109-2121Crossref PubMed Scopus (13) Google Scholar). In other studies, Raphael Salomon and colleagues were able to examine the regulatory function of the 3′-region of tobacco mosaic virus (TMV) RNA. This was accomplished by subjecting TMV RNA molecules to limited phosphorolysis by PNPase (25Salomon R. Sela I. Soreq H. Giveon D. Littauer U.Z. Enzymatic acylation of histidine to tobacco mosaic virus RNA..Virology. 1976; 71: 74-84Crossref PubMed Scopus (21) Google Scholar). Additional applications of the 3′-exonucleolytic activity of the enzyme were developed for sequence analysis of short oligoribonucleotides (26Kaufmann G. Grosfeld H. Littauer U.Z. Stepwise phosphorolysis with polynucleotide phosphorylse: a novel method for sequence analysis of oligoribonucleotides..FEBS Lett. 1973; 31: 47-52Crossref PubMed Scopus (10) Google Scholar). Finally, Gabriel Kaufmann (now Professor and Head of the Department of Biochemistry at Tel Aviv University) determined the substrate specificity of PNPase. The enzyme was found to direct the reversible addition of a single deoxynucleotidyl residue to ribooligonucleotide primers, whereas further addition of deoxynucleotidyl residues to the resulting product continued at a very slow rate (27Kaufmann G. Littauer U.Z. Deoxyadenosine diphosphate as substrate for polynucleotide phosphorylase from Escherichia coli..FEBS Lett. 1969; 4: 79-83Crossref PubMed Scopus (26) Google Scholar). The enzyme was also found to phosphorolyze aminoacyl-tRNAs, thereby yielding aminoacyl-ADP and nucleoside diphosphates (28Kaufmann G. Littauer U.Z. Phosphorolysis of aminoacyl-tRNA by polynucleotide phosphorylase from Escherichia coli..Eur. J. Biochem. 1970; 12: 85-92Crossref PubMed Scopus (11) Google Scholar). These observations prompted us to investigate the properties of ribonucleoside diphosphate analogs modified in their sugar moiety as substrates for the enzyme. We suggested that blocking of NDPs at their 3′-hydroxyl function would yield "monofunctional" substrates to which only one residue may be added to an oligonucleotide primer, thus serving as chain terminators. The blocking group can be subsequently removed chemically from the oligonucleotide products, permitting a succession of single addition reactions. This procedure was employed for the stepwise synthesis of polyribonucleotides of defined sequence (26Kaufmann G. Grosfeld H. Littauer U.Z. Stepwise phosphorolysis with polynucleotide phosphorylse: a novel method for sequence analysis of oligoribonucleotides..FEBS Lett. 1973; 31: 47-52Crossref PubMed Scopus (10) Google Scholar, 29Kaufmann G. Fridkin M. Zutra A. Littauer U.Z. Monofunctional substrates of polynucleotide phosphorylase. The monoaddition of 2′(3′)-isovaleryl-nucleoside diphosphate to an initiator oligonucleotide..Eur. J. Biochem. 1971; 24: 4-11Crossref PubMed Scopus (24) Google Scholar). Combinations of these reactions and using T4 RNA ligase to ligate the synthesized oligonucleotides allowed the synthesis of appreciable long oligonucleotides. Kaufmann further adapted the use of this ligase for unique sequence insertions and alterations in tRNA anticodon loops (30Kaufmann G. Littauer U.Z. Covalent joining of phenylalanine transfer ribonucleic acid half-molecules by T4 RNA ligase..Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3741-3745Crossref PubMed Scopus (45) Google Scholar). PNPase has been the subject of numerous studies. It was employed as a tool for producing model nucleic acids and solving many important biological problems. Thus, establishing the genetic code was facilitated by the ability of PNPase to synthesize heteropolymers and triplet nucleotides. The advances made in the understanding of the physicochemical properties of polyribonucleotide chains and their hybridization reactions, as well as the synthesis of polynucleotide inducers of interferon, are further examples of the role played by the enzyme. I was glad to have had the opportunity to write a review, together with Marianne Grunberg-Manago, summarizing the voluminous studies on this enzyme that have accumulated over the years (5Littauer U.Z. Grunberg-Manago M. Polynucleotide phosphorylase..in: Creighton T.E. Encyclopedia of Molecular Biology. Vol. 3. John Wiley and Sons, New York1999: 1911-1918Google Scholar). More recent studies in several laboratories have revealed that the PNPase gene sequence is evolutionary conserved. It is widely distributed among a variety of a
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