Carlos Spears

The interaction of transfer factor G, ribosomes, and guanosine nucleotides in the presence of fusidic acid

Abstract The reaction of factor G, ribosomes, and GTP to form a ternary complex has been investigated. The formation of the complex is associated with the hydrolysis of GTP and the amount of the resulting ribosome-G-GDP complex formed is increased in the presence of fusidic acid. In addition, in the presence of fusidic acid, a stoichiometry is observed between the amount of GDP bound in the complex and the amount of GTP hydrolyzed. The GTP analog guanylyl-5′-methylenediphosphonate (GDPCP) can also form a ternary complex with factor G and ribosomes but the formation of this complex is unaffected by fusidic acid. It is suggested that the mechanism of action of fusidic acid is to stabilize the factor G-ribosome-GDP complex and prevent its dissociation. All of the above reactions can take place on the 50S ribosomal subunit.

Regulation of the terminal reactions in methionine biosynthesis by vitamin B12 and methionine

Abstract Two of the enzymes involved in the terminal steps of methionine biosynthesis, N 5,10 -methylene-H 4 -folate reductase and the non-B 12 transmethylase, are repressed in a noncoordinate manner by both vitamin B 12 and methionine. The repressions due to vitamin B 12 and methionine are mediated by two distinct mechanisms based upon the observations obtained with three types of mutants, i.e., met J − strains (regulatory mutants), met K − strains ( S -adenosylmethionine synthetase deficient) and met H − strains (B 12 -transmethylase mutants). The repression by methionine is markedly reduced in the former two strains, whereas significant repression by vitamin B 12 is still observed. In contrast, the two enzymes are repressed by methionine in the met H − strains, whereas no repression by vitamin B 12 is observed. Furthermore, in met H + strains a relationship between B 12 transmethylase holoenzyme formation and repression has been observed. These observations indicate that the functional holoenzyme of B 12 -transmethylase is essential for the repression by vitamin B 12 . Methionine induces the non-B 12 transmethylase in all met J − strains. The repression of the non-B 12 transmethylase by vitamin B 12 in these strains is overcome by the addition of methionine to the growth medium. In addition, experiments with the S -adenosylmethionine synthetase deficient mutants suggest that S -adenosylmethionine is involved in repression of the reductase and non B 12 transmethylase.

Studies on the initiation of protein synthesis in animal tissues

Two methionine accepting tRNAs have been separated from rabbit liver tRNA by column chromatography on BD-cellulose. One of the species (Met-tRNAFMet) can be formylated using an Escherichia coli transformylase preparation, and maximal binding of this species to brain ribosomes is achieved in the presence of AUG, GTP and a 0.5 M NH4Cl extract of the ribosomes. Transferase I cannot replace the NH4Cl extract in the binding assay. The other tRNA species (Met-tRNAMet) requires GTP and transferase I for binding to ribosomes.

Studies on vitamin B12 metabolism in HeLa cells.

Abstract In the present report some aspects of vitamin B 12 metabolism in HeLa cells and other tissue culture lines have been investigated. Mangum et al . (1) previously reported the presence of higher levels of N 5 -methyl- H 4 -folate-homocysteine transmethylase in tissue culture cells grown in the presence of vitamin B 12 . The present experiments indicate that under the growth conditions used, HeLa cells do not contain sufficient vitamin B 12 to satisfy the cobamide requirement of the transmethylase, and the increase in enzymatic activity that is observed when the cells are grown in the presence of vitamin B 12 results from the conversion of the apoenzyme form of the enzyme to active holoenzyme. No effect of the vitamin on the oxidation of propionate to CO 2 was observed in these cells suggesting that the amount of 5′-deoxyadenosyl B 12 (DBCC) present was sufficient so that the methylmalonyl CoA isomerase reaction was not rate limiting. Extracts of HeLa cells readily convert hydroxy-B 12 to DBCC in the presence of ATP and a reducing system. The optimum conditions for the enzymatic synthesis of DBCC are described.

The mRNA-directed synthesis of the α-peptide of β-galactosidase, ribosomal proteins L12 and L10, and elongation factor Tu, using purified translational factors

Abstract A crude messenger-RNA preparation capable of directing the synthesis of the α-peptide of β-galactosidase, ribosomal proteins L12 and L10, and elongation factor Tu (EF-Tu) has been obtained from a β-galactosidase overproducing strain, Escherichia coli A324-5. The in vitro synthesis of these proteins has been investigated using either a partially fractionated Escherichia coli extract or purified translational factors. The mRNA-directed synthesis was blocked by translational inhibitors such as chloramphenicol and puromycin, but not by transcriptional inhibitors such as rifampicin and actinomycin D. Furthermore, the mRNA-directed synthesis of the α-peptide of β-galactosidase was not affected by adenosine 3′,5′-cyclic monophosphate, isopropyl-β- d -thiogalactoside, and guanosine 5′-diphosphate-3′-diphosphate. In a more defined system using 30 highly purified components, greater than 50% of the protein synthesizing activity seen with the crude extracts has been obtained. With this in vitro system, it has been possible to obtain good dependencies for protein synthesis on the three initiation factors, EF-Tu, the group of 20 aminoacyl-tRNA synthetases, and ribosomes. N10-Formyl-H4folate-Met-tRNAf transformylase and EFG gave only a twofold or less stimulation, due very likely to contamination of these proteins in other factors. As yet, no requirement for EF-Ts, release factors, or ribosome release factor has been obtained.

Studies on the in vitro synthesis of β-galactosidase: Necessary components in the ribosomal wash

Abstract The factors present in the NH 4 Cl wash of ribosomes that are required for the DNA directed in vitro synthesis of β-galactosidase have been examined in a system dependent on washed Escherichia coli ribosomes, the NH 4 Cl wash of ribosomes, and a 200,000 g supernatant fraction. Approximately 40% of the activity present in the wash was recovered by substituting purified initiation factors 1 and 3 for the crude ribosomal wash. There was little or no effect of initiation factor 2, because of significant amounts of this factor in the supernatant fraction. Full activity initially present in the wash could be restored by the addition to the initiation factors of a protein fraction present in the wash. This protein fraction has been separated from initiation factors but its function is still not known. With this system, it was possible to show that the initiation factors also stimulate the transcription of the lac operon or protect the lac mRNA once formed.

Studies on the in vitro transcription and translation of the lac operon

With the use of rifampicin to stop the initiation of RNA synthesis, the in vitro synthesis of β -galactosidase has been carried out in two separate stages, transcription and translation. In this system it has been possible to examine whether substances, which affect the synthesis of the enzyme, function at the transcription or translation level of protein synthesis. Guanosine 5′-diphosphate 2′ (or 3′) diphosphate and 3′5′ cyclic AMP, affect mainly the transcription of the lac operon, whereas polyethyleneglycol 6000 stimulates the translation of lac mRNA or protects the mRNA. It appears that unknown factors present in the 1 m NH 4 Cl ribosomal wash are essential for transcription of the lac operon even in the presence of saturating levels of RNA polymerase. Both the S-200 fraction and washed ribosomes stimulate transcription of the lac operon as do 30S and 50S ribosomal subunits. The effect of the 50S particles can be replaced by the 50S ribosomal proteins, L 7 and L 12 .

Further studies on the cell-free synthesis of procollagen-collagen by chick embryo polysomes

Abstract Free and membrane bound polysomes were prepared from 8-day-old chick embryos. Both polysome preparations were equally active in protein synthesis but procollagen-collagen synthesis was carried out exclusively by the membrane bound polysomes. The collagenous product was analyzed by DEAE-cellulose chromatography and after hydroxylation with peptidyl proline hydroxylase had a hydroxyproline/proline ratio of 0.77. This suggests that the collagenous product synthesized by the membrane bound polysomes is procollagen.

Activity of different forms of initiation factor 2 in the in vitro synthesis of β-galactosidase

Abstract Two forms of initiation factor 2, (IF-2α, Mr, 118,000 and IF-2β, Mr 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.

DNA-directed in vitro synthesis of β-galactosidase: Dependencies on elongation factor Tu and tRNA

Abstract The DNA-directed in vitro synthesis of β-galactosidase has been investigated in a system dependent on ribosomes, a salt wash of the ribosomes and two DEAE eluates (0.25 m and 1 m KPO 4 ) of the supernatant fraction. Fractionation of the 0.25 m KPO 4 DEAE eluate has made it possible to obtain elongation factor Tu (EFTu) and exogenous tRNA dependencies for β-galactosidase synthesis. The requirement for EFTu was related to the amount of tRNA present in the system. Excess amounts of tRNA caused a marked inhibition of enzyme synthesis which could, in part, be prevented when additional EFTu was added.