Severo Ochoa

Enzymic synthesis of polynucleotides I. polynucleotide phosphorylase of Azotobacter vinelandii

The isolation, partial purification and some properties of polynucleotide phosphorylase of Azotobacter vinelandii are described. The enzyme catalyzes the synthesis of highly polymerized ribonucleic acid-like polynucleotides from 5′-nucleoside diphosphates with release of orthophosphate. The reaction requires magnesium ions and is reversible. Thus, the enzyme also catalyzes the phosphorolysis of polynucleotides to yield the corresponding 5′-nucleoside diphosphates. The preparation and isolation of a number of polynucleotides containing one or several kinds of mononucleotide units is described. The scope, mechanism and significance of the reaction are discussed.

Enzymes of fatty acid metabolism

Abstract The intermediates in the biological breakdown and synthesis of fatty acids are S-acyl derivatives of coenzyme A. Fatty acid synthesis is accomplished through repetition of a cycle of four consecutive reactions: a. Condensation of two molecules of acetyl CoA to form acetoacetyl CoA and coenzyme A (CoASH); b. reduction of acetoacetyl CoA to β-hydroxybutyryl CoA; c. dehydration of β-hydroxybutyryl CoA to crotonyl CoA, and d. reduction of crotonyl CoA to butyryl CoA. A new cycle is started by the reaction of butyryl CoA with another molecule of acetyl CoA to form β-keto-caproyl CoA + CoA-SH, and so forth. The cycle is repeated eight times until stearyl CoA is formed. All four reactions of the fatty acid cycle are reversible and fatty acid oxidation, once the fatty acid is activated through conversion to the corresponding S-acyl CoA derivative, proceeds by a reversal of the above sequence. There are two main mechanisms for activation of fatty acids: (a) By a reaction with ATP and CoA to form S-acyl CoA, adenosine monophosphate and pyrophosphate, and (b) by transfer of CoA from certain acyl CoA compounds such as acetyl CoA or succinyl CoA. The isolation and identification of some of the key enzymes of fatty acid metabolism is outlined and their mechanism of action discussed.

Translation of the genetic message: VII. Role of initiation factors in formation of the chain initiation complex with Escherichia coli ribosomes☆

Abstract Washing of Escherichia coli Q13 ribosomes with 1.0 m ammonium chloride yields, in addition to the known initiation factors F 1 and F 2 , a third factor F 3 . All three factors are required for translation of natural messenger RNA (mRNA), e.g., phage RNA, in the cell-free E. coli system. They function in formation of the polypeptide chain initiation complex. Incubation of purified ribosomes with 32 P-labeled Qβ phage RNA and methionine- 14 C-labeled formylmethionyl-transfer RNA, at low magnesium ion concentrations, followed by zonal centrifugation analysis in sucrose gradients, showed that formation of the initiation complex involves at least two steps: (1) The F 3 -dependent binding of mRNA to the ribosome, and (2) the F 2 -dependent binding of formylmethionyl-transfer RNA to the mRNA-ribosome complex to form the initiation complex proper.

“Fluorokinase” and pyruvic kinase

Abstract 1. 1. The enzyme catalyzing the CO2-dependent phosphorylation of fluoride by adenosine triphosphate to yield monofluorophosphate, an activity referred to as “fluorokinase,” has been isolated in crystalline form from rabbit muscle extracts. The crystalline enzyme has been found to possess considerable pyruvic kinase activity. 2. 2. A number of observations including (a) constancy of pyruvic kinase to “fluorokinase” activity ratio throughout purification and on repeated recrystallization; (b) similar ratio of the two activities for preparations purified by different methods either for pyruvic kinase or for “fluorokinase” activity; (c) requirement of K+ and same broad nucleotide specificity for the two activities (reaction with the nucleoside di- or triphosphates of adenosine, guanosine, uridine, cytidine, and inosine); and (d) inhibition of fluorophosphate formation by phosphoenolpyruvate and by pyruvate, suggest that both the pyruvic kinase and “fluorokinase” reactions are catalyzed by one and the same protein. 3. 3. The possible mechanism of the essentially irreversible “fluorokinase” reaction is discussed.

Complexing ability and coding properties of synthetic polynucleotides

Polyribothymidylic acid promotes incorporation of phenylalanine into acid-insoluble products in a cell-free Escherichia coli system. Under conditions in which poly U‡ exists as a random coil, poly rT possesses considerable ordered structure. The structural differences between the two polymers are reflected in their respective effects on phenylalanine incorporation at different temperatures. At 20°C poly rT is much less effective than poly U, probably because its ordered structure interferes with ribosomal attachment. At 45°C, when it exists as a random coil, poly rT promotes phenylalanine incorporation at a higher initial rate than does poly U. This may be ascribed to a higher complexing ability of the former polymer, facilitating its interaction with the adaptors of phenylalanine transfer RNA. The effect of temperature and Mg 2+ concentration, factors that affect interaction between poly- and oligonucleotides, on the promotion of incorporation of leucine and phenylalanine by poly U was examined. This apparent “ambiguity” becomes more pronounced with conditions that increase the complexing ability of the polymers. It is higher with poly rT than with poly U and increases in either case with decreased temperature or increased Mg 2+ concentration. These conditions also lead to significant incorporation of amino acids (isoleucine, serine, tyrosine) that, like leucine, have two TPs in their code triplets. With low Mg 2+ (0·01 M ), or in the presence of excess unlabeled phenylalanine and transfer RNA, poly U promotes the incorporation of phenylalanine but not that of leucine, i.e. the “ambiguity” disappears. Phenylalanine is a strong competitor of the incorporation of leucine promoted by poly U and related polynucleotides. Polyadenylic acid becomes more effective for polylysine synthesis as the temperature is raised from 20 to 45°C and the Mg 2+ concentration is slightly increased. Facilitation of ribosomal attachment through partial disorganization of the secondary structure of this polynucleotide may be largely responsible for this effect.

Synthesis of virus-specific proteins in Escherichia coli infected with the RNA bacteriophage MS2.

The synthesis of virus-specific proteins following infection of Escherichia coli with the RNA phage MS2 was studied using spheroplasts exposed to low concentrations of actinomycin D in the presence of an amino acid mixture containing radioactive leucine or histidine. Under the conditions employed there was about 75% inhibition of virus production and 99% inhibition of host protein synthesis by actinomycin. Fractionation of the phage-induced proteins by acrylamide gel electrophoresis revealed the presence of three major peaks, I, II, and III. The largest one (peak III) was characterized as coat protein. The rate of synthesis of proteins I and II declines before that of the coat protein has reached its maximum.

Partial purification of isocitric dehydrogenase and oxalosuccinic carboxylase

Abstract Partial purification of the isocitric dehydrogenase and oxalosuccinic carboxylase activities of pig heart has been obtained by means of ammonium sulphate and ethanol fractionation of an acetone powder extract. The purification reached was about six-fold with a yield of about 15%. No separation of the two activities has thus far been accomplished. The strong inhibition of oxalosuccinic carboxylase activity by isocitric acid has been confirmed using an optical test system.

Eucaryotic oligonucleotides affecting mRNA translation

Abstract High speed-supernatants and ribosomal salt washes of dormant and developing Artemia salina embryos contain a potent inhibitor of translation; it blocks the elongation factor EF-1-dependent ribosomal binding of aminoacyl-tRNA. A translation activator that counteracts the effect of the inhibitor is found in the same fractions from developing embryos; there is little activator in undeveloped cysts. The appearance of the activator may be responsible for the onset of protein synthesis when development resumes. Both compounds are oligonucleotides. The inhibitor, M r about 6000, is rich in pyrimidines (47% U, 11% A, 26% C, 16% G), sensitive to RNase A, and resistant to RNase T1. The activator, M r about 9000, is rich in guanine (33% U, 10% A, 6% C, 51% G), sensitive to RNase T1, and resistant to RNase A. It complexes with the inhibitor and inactivates it. Inhibitor and activator seem to be end products of hydrolysis of embryo RNA by RNase T1 and RNase A, respectively, and ribosomal salt washes of developing embryos have higher RNase A activity than corresponding fractions from dormant cysts.

Initiation of protein synthesis.

Protein synthesis is the last step in expression of genetic information. In this step, messenger RNA is translated to yield a polypeptide chain whose amino acid sequence is determined by the nucleotide sequence of the messenger. There are three steps in translation: initiation, elongation, and termination. Chain initiation and the beginning of chain elongation are schematically represented in Figure 1. In prokaryotes the mRNA is first bound to the small ribosomal subunit. This is followed (step 1) by binding of the initiator aminoacyl tRNA to the small subunit-mRNA complex. This step requires Mg 2+, initiation factors (IF), and GTP. Next, the larger ribosomal subunit joins this complex to form the initiation complex proper (step 2) and the stage is set for chain elongation. The second aminoacyl-tRNA is bound in a reaction (step 3) that requires GTP and an elongation factor (EF-1) whereby GTP is hydrolyzed to GDP and Pi. The first (initiator) and second aminoacyl-tRNA occupy sites on the ribosome designated as the peptidyl (P) and aminoacyl (A) site, respectively. Peptide bond formation takes place (reaction 4); the A site now bears dipeptidyl-tRNA while the P site bears the deacylated initiator tRNA. In the next (translocation) step (step 5)the ribosome is displaced relative to the messenger-peptidyl tRNA complex; the initiator tRNA is ejected and the peptidyl tRNA is now on the P site. This sets the stage for binding of the third aminoacyl tRNA and these processes are repeated until the polypeptide chain is completed. Translocation requires a second elongation factor (EF-2) and one more GTP which is hydrolyzed to GDP and Pi. Whereas the steps and mechanisms of protein syn-

[1] Polynucleotide phosphorylase from Azotobacter vinelandii nXDP ⇄ (XMP)n + nPi

Publisher Summary This chapter describes the assay and purification of polynucleotide phosphorylase from Azotobacter vinelandii . Two methods are used for tile assay of polynucleotide phosphorylase. In one, the rate of exchange of P i 32 with ADP is measured, and in the other, the rate of formation of ADP resulting from the phosphorolysis of polyadenylic acid is determined spectrophotometrically by coupling with the reactions catalyzed by pyruvic kinase and lactic dehydrogenase. A freshly prepared 2% solution of protamine sulfate is added dropwise to 41 ml of the dialyzed eluate at 0° with mechanical stirring. The enzyme has been purified 500-fold and appears to be essentially devoid of nuclease activity. It is found that when assayed viscosimetrically with polyuridylic acid as substrate, 7 μg. of enzyme brought about a decrease in viscosity at about the same rate as 0.01 μg. of crystalline pancreatic ribonuclease. The purified enzyme contains small amounts of a firmly bound oligoribonucleotide. It is observed that although preparations of Azotobacter polynucleotide phosphorylase react with individual ribonucleoside 5′-diphosphates to give the corresponding homopolymers or with mixtures of ribonucleoside diphosphates to form different copolymers, a single enzyme seems to be involved.