Joanne M. Ravel

Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride

Abstract A procedure is described for radiolabeling proteins in vitro by reductive alkylation. The proteins are treated with [ 14 C]formaldehyde in the presence of sodium cyanoborohydride, a reducing agent that is stable in aqueous solution at pH 7. The advantage of this procedure is that the reaction can be carried out at neutral pH for extended periods of time and over a wide range of temperatures (0–37°C). Moreover, owing to the flexibility in the reaction conditions afferded by the use of sodium cyanoborohydride, higher incorporation of radiolabel into protein can be attalned.

Evidence for a guanine nucleotide-aminoacyl-RNA complex as an intermediate in the enzymatic transfer of aminoacyl-RNA to ribosomes

Abstract In recent investigations in this laboratory (1,2) two fractions required for polyuridylic acid-directed synthesis of polyphenylalanine were obtained from extracts of Escherichia coli W by chromatography on DEAE-Sephadex. One of the fractions, designated F-I, catalyzes the binding of aminoacyl-RNA to ribosomes in the presence of mRNA and GTP. This fraction has a high affinity for GTP and contains a GTPase that is stimulated by aminoacyl-RNA but not by stripped sRNA. Recently, Allende et al. (3) have shown that an enzyme fraction from E. coli B, which appears to be comparable to F-I, binds 3H-GTP. In the present investigation 3H-GTP was also found to interact with F-I to form a complex that is retained on a Millipore filter; however, if γ-32P-GTP is used, very little radioactivity is retained by the filter. On further investigation the amount of 3H-labeled compound bound to the protein was found to be decreased by the addition of aminocyl-RNA but not by stripped sRNA nor by N-acetylaminoacyl-RNA. Preliminary data indicate that F-I catalyzes the formation of a guanine nucleotide-aminoacyl-RNA complex which may be the intermediate product formed in the “enzymatic” transfer of aminoacyl-RNA to ribosomes.

A study of the enzymic transfer of aminoacyl-RNA to Escherichia coli ribosomes.

Abstract Three fractions, F-IA, F-IB, and F-II, have been obtained from extracts of Es- cherichia coli W by chromatography on DEAE-Sephadex. The guanosine 5′-triphosphate (GTP)-dependent transfer of aminoacyl-RNA to ribosomes is catalyzed by either F-IA or F-IB, and polypeptide synthesis is catalyzed by either fraction in the presence of F-II. Fraction F-IB is more labile than F-IA. Both F-IA and F-IB interact with GTP to form guanine nucleotide-enzyme complexes that are retained by Millipore filters. In the presence of aminoacyl-RNA, F-IA and F-IB interact with GTP to form a complex that is not retained by a Millipore filter. Guanosine 5′-diphosphate (GDP) also interacts with F-IA or F-IB to form a complex; however, no subsequent interaction with aminoacyl-RNA is observed. Both the GTP-dependent enzymic transfer of aminoacyl-RNA to ribosomes and the interaction of enzyme with GTP are inhibited by GDP. The enzymic transfer of aminoacyl-RNA to ribosomes is stimulated by NH 4 + and a sulfhydryl compound, and the interaction of the guanine nucleotide-enzyme complex with aminoacyl-RNA is also stimulated by NH 4 + . Deacylated sRNA, which inhibits nonenzymic binding, has no significant effect on the enzymic binding of aminoacyl-RNA to ribosomes. The guanine nucleotide-aminoacyl-RNA complex formed by the interaction of GTP, phenylalanyl-RNA, and either F-IA, F-IB, or a mixture of the two can be recovered by filtration through a Millipore filter and chromatography on Sephadex G-25. The active fractions from the Sephadex G-25 column contain close to stoichiometric amounts of phenylalanine and the guanine moiety and the -γ-phosphate moiety of the GTP. The amount of phenylalanine transferred to the ribosomes from the complex is 2-fold greater than the amount transferred from phenylalanyl-RNA alone and is equivalent to the amount transferred from phenylalanyl-RNA in the presence of enzyme and GTP. The guanine moiety of the complex is also transferred to the ribosomes, but no significant transfer of the γ-phosphate of the GTP present in the active fractions is observed.

[32] GTP-dependent binding of aminoacyl-tRNA to Escherichia coli Ribosomes

Publisher Summary This chapter describes the general procedures for preparation of ribosomes, [ 14 C]- or [3H]Phenylalanyl-tRNA, N-Acetyl-[ 14 C]phenylalanyl-tRNA, separation of TI s , TI u , and TII by DEAE-Sephadex chromatography, preparation of complex H, and binding of phenylalanyl-tRNA to ribosome.poly(U) complexes. Two transfer factors, TI s and Tl u , and GTP are required for the binding of aminoacyl-tRNA at the acceptor site of a ribosome-mRNA complex. Binding of [ 14 C]- or [ 3 H]aminoacyl-tRNA to the ribosomes is conveniently measured by adsorption of the ribosomal complex on a nitrocellulose (Millipore) filter. In the presence of aminoacyl-tRNA, TI s and TI u interact with GTP to form an aminoacyl-tRNA TI u GTP complex (complex II) that is not retained by a Millipore filter; TI s is still retained by the filter under these conditions. The binding of the phenylalanyl-tRNA moiety of complex II (prepared with [ 14 C]phenylalanyl-tRNA and a mixture of [ 3 H]GTP and [γ- 32 P]GTP) to a ribosome.poly (U) complex occurs at low concentrations of Mg 2+ and at low temperatures. When the phenylalanyl-tRNA moiety of complex II is bound at the acceptor site of a ribosome poly (U) complex carrying N-acetylphenylalanyl-tRNA at the donor site, peptide bond formation occurs.

Purification and properties of a Met-tRNAf binding factor from wheat germ.

Abstract The 40–60% ammonium sulfate fraction of the postribosomal supernatant of wheat germ catalyzes the binding of Met-tRNA f to 40 S ribosomal subunits, and in addition, interacts with Met-tRNA f , in the absence of 40 S ribosomal subunits and Mg 2+ to form a complex that is retained on a Millipore filter. Upon chromatography on diethylaminoethyl (DEAE)-cellulose, two fractions having this latter activity were obtained, a 0.05 m KCl fraction and a 0.12 m KCl fraction. The 0.12 m KCl fraction, but not the 0.05 m KCl fraction, also contained the factor that catalyzes the binding of Met-tRNA f to 40 S ribosomal subunits. When the 0.12 m KCl fraction from DEAE-cellulose was subjected to chromatography on Sephadex G-200 and on phosphocellulose, it was found that both activities copurified throughout these procedures, and both were purified more than 300-fold. In addition, both activities have similar heat-inactivation profiles. The formation of a complex between the factor and Met-tRNA f in the absence of 40 S subunits is stimulated three- to fourfold by GTP and is inhibited by GDP. Ternary complex formation is specific for Met-tRNA f and is decreased in the presence of Mg 2+ . The binding of Met-tRNA f to 40 S subunits is stimulated three- to fourfold by AUG, and when AUG is present, omission of GTP reduces the amount of Met-tRNA f bound by only about 30%. The factor catalyzes the binding of Met-tRNA m to 40 S subunits about one-fifth as well as Met-tRNA f and catalyzes the poly(U)-directed binding of Phe-tRNA by about 50% as well. Upon further investigation, it was found that the binding of Met-tRNA f to 40 S subunits that occurs in the absence of template is GTP dependent, being reduced more than 90% by the omission of GTP. No detectable binding of Phe-tRNA to 40 S subunits is observed in the absence of poly(U), indicating that template-independent binding is specific for Met-tRNA f . Both ternary complex formation and template-independent binding of Met-tRNA f to 40 S subunits are reduced more than 90% by treatment of the enzyme with N -ethylmaleimide. However, binding of Met-tRNA f to 40 S subunits in the presence of AUG is not affected by treatment of the enzyme with N -ethylmaleimide. The results of this investigation suggest that in wheat germ, the Met-tRNA f binding activities described above may reside in a single oligomeric protein.

A general involvement of acceptor ribonucleic acid in the initial activation step of glutamic acid and glutamine

Abstract The glutamyl- and glutaminyl-ribonucleic acid (RNA) synthetases have been partially purified from yeast and pork liver, and the amino acid-dependent adenosine triphosphate-pyrophosphate exchange reactions catalyzed by these enzymes have been studied. Analogous to the results obtained previously with the glutamyl- and glutaminyl-RNA synthetases from Escherichia coli , the glutamyl-RNA synthetases from yeast and pork liver were found to catalyze the glutamate-dependent exchange reaction only at high concentrations of glutamate in the absence of acceptor RNA. The addition of acceptor RNA greatly decreases the concentration of glutamic acid necessary to promote the exchange reaction. With the glutaminyl-RNA synthetases from yeast and pork liver, no detectable exchange reaction is observed except in the presence of acceptor RNA. From these data, it appears that there is a general involvement of acceptor RNA in the initial activation step of glutamic acid and glutamine.

The effects of phenylalanine and tyrosine analogs on the synthesis and activity of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthetases

Abstract Two 3-deoxy- d - arabino -heptulosonic acid 7-phosphate (DAHP) synthetases, one of which is inhibited by tyrosine and the other by phenylalanine, exist in Escherichia coli mutant 83-24. The abilities of a number of analogs of phenylalanine and tyrosine to substitute for the natural metabolites in preventing the synthesis of or inhibiting the activity of the two DAHP synthetases have been examined. It can be concluded that the enzymatic sites to which phenylalanine and tyrosine must conform on the respective DAHP synthetases are relatively specific. An unsubstituted 4-position of the aromatic ring enhances binding of an analog in place of phenylalanine, and for effective binding in lieu of tyrosine, a 4-hydroxyl group greatly increases binding affinity of an analog. For prevention of enzyme synthesis, a variety of analogs are appreciably active, indicating that the structural requirements for repression are somewhat less specific than the requirements for regulation of the activity of the DAHP synthetases. Some effects of these inhibitors have been correlated with their ability to prevent growth of the parent organism, Escherichia coli W.

Evidence for activities of rabbit reticulocyte elongation factor 1 analogous to bacterial factors EF-Ts and EF-Tu

Abstract Preparations have been obtained from rabbit reticulocyte elongation factor 1 (EF-1) that exhibit activities analogous to the heat stable and heat labile factors, EF-Ts and EF-Tu, of Escherichia coli . The heat stable fraction, prepared by heating EF-1 in the presence of GTP, has virtually no activity in poly (U)-directed polyphenylalanine synthesis. The fraction exhibiting activity similar to bacterial EF-Tu is obtained by the interaction of EF-1 with GTP and phenylalanyl-tRNA followed by passage of the solution through a nitrocellulose filter. The filtrate, which alone has low activity in polyphenylalanine synthesis, when combined with the heat stable fraction gives high activity suggesting that the heat stable preparation catalyzes recycling of the filtrate component.

Initiation factor 3 requirement for the formation of initiation complexes with synthetic oligonucleotides

Abstract Initiation factor IF-3 is required for the poly (U)-directed binding of N-acetyl-Phe-tRNA to 70S ribosomes as well as for the binding of fMet-tRNA directed by poly (U,G), AUG, and bacteriophage f 2 RNA. The formation of the 70S initiation complex is dependent upon IF-2 and is stimulated by IF-1. The requirement for IF-3 is not alleviated by high concentrations of the synthetic templates.

Some inhibitory interrelationships among leucine, isoleucine and valine

Abstract The effects of varying concentrations of leucine, isoleucine, and valine on the growth of Leuconostoc dextranicum 8086 indicate that each of these amino acids inhibits the utilization of the other two amino acids by this organism. While leucine and isoleucine exert synergistic effects in inhibiting the utilization of valine, the other two pairs of amino acids probably exert only additive inhibitory effects. The corresponding keto acid or a peptide of the amino acid in some cases is more effective than the amino acid in reversing inhibitions involving these three amino acids in this organism or in Lactobacillus arabinosus .