DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors

The phage DNA-directed synthesis of beta-galactosidase has been examined in a system containing the following purified Escherichia coli factors: RNA polymerase; cyclic AMP receptor protein; N10-formyltetrahydrofolate Met-tRNAf transformylase; initiation factors 1, 2, and 3; elongation factors Tu, Ts, and G; release factors 1 and 2; 20 aminoacyl-tRNA synthetases; L factor (Kung, H. F., Spears, C., and Weissbach, H. (1974) J. Biol. Chem. 250, 1556-1562); and Lalpha (Kung, H.-F., Spears, C., and Weissbach, H. (1976) Fed. proc. 35, 1537). Under these conditions, beta-galactosidase synthesis occurs at less than 1% of the rate obtained with unfractionated extracts, which suggested that other required components were lacking. The difficulty in obtaining large amounts of the purified aminoacyl-tRNA synthetases for these studies made it necessary to modify the system. It was possible to conserve many of the purified aminoacyl-tRNA synthetases since at least 13 of them could be replaced by an Ehrlich ascites extract. The ascites extract plus other E. coli purified factors was used as a basic system to search for additional components required for beta-galactosidase synthesis. The present report describes the purification from E. coli extracts of three fractions, called Lbeta, Lgamma, and Ldelta, that are needed to restore enzyme synthesis.     

Dependence of mitochondrial protein synthesis initiation on formylation of the initiator methionyl-tRNAf.

The effect of N10-formyl-H4folate on mitochondrial peptide chain initiation has been studied in isolated mitochondria of Saccharomyces cerevisiae. The addition of N10-formyl-H4-folate strongly stimulates the incorporation of amino acids into mitochondrial protein at both 6 and 15 mm Mg2+. Still higher stimulation (up to 10-fold) has been obtained in the production of de novo synthesized initial peptides, measured as peptidyl puromycin derivatives. The maximum effect is observed at 0.1 mM N10-formyl-H4folate. At 5 mM puromycin, the ratio formylated/unformylated peptides is 3, as shown by electrophoretic analysis. At 10 mM puromycin, the ratio is increased to more than 6. This is due to the presence of deformylase and amidohydrolase activities, which are more effective the longer the initial peptide is synthesized; at increasing puromycin concentrations, progressively shorter peptide chains are formed. Chemically synthesized fMet-puromycin and Met-puromycin are virtually stable when incubated with intact or frozen and thawed mitochondria. More careful kinetic analysis shows an early cessation of the initial peptide formation in the samples without N10-formyl-H4-folate. This indicates that the formylation of methionyl-tRNA formylatable species is an absolute requirement for mitochondrial peptide chain initiation.     

DNA-directed in vitro synthesis of beta-galactosidase. Purification and characterization of stimulatory factors in an ascites extract.

Previous studies have described a partially defined system for the DNA-directed in vitro synthesis of beta-galactosidase (Kung, H.F., Redfield, B., Treadwell, B.V., Eskin, B., Spears, C., and Weissbach, H. (1977) J. Biol. Chem. 252, 6889-6894). An Ehrlich ascites extract was shown in these in vitro studies to acylate Escherichia coli tRNA with 13 amino acids, and the ascites extract was used in place of the corresponding 13 E. coli aminoacyl-tRNA synthetases. The present studies indicate that the ascites extract is supplying an additional protein factor, besides the aminoacyl-tRNA synthetases, that stimulates the DNA-directed synthesis of beta-galactosidase. The protein factor has been highly purified and may be functioning by protecting mRNA against degradation. In addition, NAD or T4 DNA ligase stimulates the synthesis of beta-galactosidase in the partially defined system.     

DNA-directed in vitro synthesis of Escherichia coli beta-isopropylmalate dehydrogenase.

The in vitro synthesis of beta-isopropylmalate dehydrogenase (EC 1.1.1.85), an enzyme involved in leucine biosynthesis, has been obtained using as template DNA from the hybrid plasmid (pLC1) which contains the Escherichia coli leucine operon. Enzyme synthesis in vitro is stimulated about 2-fold by guanosine-5'-diphosphate-3'-diphosphate and inhibited about 60% by 2 X 10(-4) M L-leucine.     

In vitro polyoma DNA synthesis: requirement for cytoplasmic factors.

Purified nuclei from polyoma-infected mouse (3T3) cells were found to be greatly reduced in their ability to synthesize viral DNA in vitro when compared with a crude system consisting of an unfractionated hypotonic lysate of the infected cells. The synthetic capacity of the nuclei could be fully reconstituted when a high-speed cytoplasmic supernatant was added back to them. Cytosols from uninfected mouse, monkey, and hamster cells were equally as effective in stimulating purified nuclei as that of virus-infected mouse cells. Optimal complementation required high concentrations of the cytosol, and most of the complementing activity was destroyed by heating to 60 C. Dialysis had no effect on the activity. Analysis of the viral DNA synthesized in purified nuclei showed an accumulation of Okazaki-type short DNA chains, which could be chased into viral progeny DNA strands if cytosol was added back to the nuclei. Kinetic analysis of the pulse-labeling pattern of viral replicative DNA showed a strong dependence of the extension of viral progeny strands and of the processing of Okazaki-type fragments on the amount of cytosol present during the reaction. It is suggested that the cytoplasmic DNA polymerase might be one of the active components in the cytosol, but most likely not the only one.     

Bacteriophage M13 DNA-directed in vitro synthesis of gene 5 protein

A method is presented which allows one to follow in a relative rapid and accurate way the synthesis of gene 5 protein of the bacteriophage M13 in a DNA-dependent cell-free system. Due to its unique property of binding selectively to single-stranded but not to double-stranded DNA, gene 5 protein can readily be separated, by means of sucrose densitygradient centrifugation, from the other polypeptides synthesized in the cell-free system. The meaning of this technique for the elucidation of the mechanism(s) which regulates gene 5 protein synthesis is discussed.     

Mutant forms of beta-galactosidase with an altered requirement for magnesium ions.

By random approaches we have previously isolated many variants of Escherichia coli beta-galactosidase within a short contiguous tract near the N-terminus (residues 8-12 of wild-type enzyme), some of which have increased stability towards heat and denaturants. The activity of these mutants was originally analysed and quantitated in situ in activity gels without the addition of magnesium ions to the buffer system. We now show that the improved stability is only observable under such conditions of limiting magnesium ion concentrations or in the presence of appropriate concentrations of a metal chelator. In the presence of EDTA, purified preparations of one of these mutant enzymes were much more resistant to denaturants than wild-type, but this differential was completely nullified in the presence of 1 mM Mg2+. However, the stability of this mutant enzyme in EDTA was lower than that shown by it, or the wild-type enzyme, in the presence of magnesium ions. In addition, certain alterations within another N-terminal tract (residues 27-31 of wild-type) resulted in enzymes with greater dependence on Mg2+ than natural beta-galactosidase. We conclude that a small number of residue changes in a large protein can profoundly modulate the requirement for metal ion stabilization, allowing partial abrogation of this need in certain cases. Thus, some enzymes which require divalent metal ions for structural purposes only may be engineered towards metal independence.     

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

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 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.     

Metabolic regulation of beta-galactosidase synthesis in Escherichia coli. A test for constitutive ribosome synthesis.

The rate of differential synthesis of beta-galactosidase (alphalac) was measured in maximally induced cultures of Escherichia coli B/r with 0.01 M-inducer and 0.01 M-cyclic AMP. The value of alphalac decreases with growth rate (60% between 0.67 and 2.1 doublings/h) and after a nutritional shift-up. This decrease is presumed to reflect a decrease in the intracellular concentration of free active RNA polymerase after a shift-up, which implies that the increase in ribosome synthesis after a shift-up is due to an active induction of the ribosomal components.     

Catecholamine requirement for hamster sperm motility in vitro.

Homogenates of hamster and bovine glands contain a "sperm motility factor" (SMF) that stimulates the motility of hamster epididymal spermatozoa in vitro. The potency of these adrenal preparations was severely attenuated after gel filtration on a Sephadex G-10 column. This loss of activity was ascribed to the retardation and separation of co-factors for SMF which appeared to be catecholamines. The sperm motility-stimulating activity of the SMF-containing fractions was fully restored by addition of either the 'retarded' fractions or catecholamines (epinephrine or norepinephrine). Neither the catecholamines nor the 'retarded' fractions were able to sustain vigorous sperm motility in the absence of the SMF-containing fractions. The potentiating action of catecholamines on SMF was mimicked by the adrenergic agonists isoproterenol and phenylephrine and inhibited by the alpha-adrenergic antagonist phentolamine, but not by the beta-adrenergic antagonist propranolol. Our results indicate that one or more catecholamines are essential co-factors of SMF and demonstrate that hamster spermatozoa require catecholamines for their motility in vitro.     

Flagellar synthesis in Salmonella typhimurium: requirement for ribonucleic acid synthesis

The micro-complement-fixation assay has been demonstrated to be a sensitive assay for flagella which occur in nanogram amounts. By use of this assay, it was found that flagellar synthesis occurs during starvation of Salmonella typhimurium for tryptophan, an amino acid not present in flagellar protein. Under these conditions net ribonucleic acid (RNA) synthesis was reduced to approximately 10% of the control rate. Less than 1 mug of actinomycin D per ml further reduced RNA synthesis to less than 1% of the control rate in a culture sensitized by prior treatment for 5 min at 37 C with 5 x 10(-4)m ethylenediaminetetraacetate in 0.33 m tris(hydroxymethyl)aminomethane-chloride (pH 8.0). In the presence of actinomycin D, no synthesis of flagellar protein could be detected. Analysis of fractions of RNA separated by zone centrifugation indicated that actinomycin D reduces the production of template RNA as well as of ribosomal RNA. This suggests that in S. typhimurium the production of flagellar protein requires the concomitant synthesis of RNA. There is no evidence that a stable messenger RNA specific for flagellar synthesis is present.