Sandra R. Lax

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.

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.

Interaction of rabbit reticulocyte elongation factor 1 with guanosine-triphosphate and aminoacyl-transfer ribonucleic acid

Abstract Elongation Factor 1 (EF-1) from rabbit reticulocytes interacts with GTP to form a complex that is retained on a nitrocellulose filter. EF-1 also interacts with GDP; however, the concentration of GDP required for maximal complex formation is higher than the concentration of GTP required and the extent of binding is lower. Interaction of EF-1 with GTP in the presence of various aminoacyl-tRNAs from rabbit liver or E. coli results in a 50–75% decrease in the amount of GTP complex retained on a filter. No reduction in the amount of GTP complex retained is observed with deacylated tRNA or with N -acetylphenylalanyl-tRNA. EF-1 is inactivated by heating at 37 °C in the presence of GTP. Aminoacyl-tRNA protects EF-1 from the inactivation observed in the presence of GTP. These data indicate that an interaction of reticulocyte EF-1 with GTP and aminoacyl-tRNA occurs; however, attempts to demonstrate the formation of a stable ternary complex by chromatography on Sephadex G-150 were unsuccessful. Also, no difference is observed between the rate of binding of aminoacyl-tRNA to reticulocyte ribosomes obtained with EF-1 and the rate obtained with EF-1 that had been incubated previously with GTP and aminoacyltRNA.