Relief of polarity in DNA-dependent cell-free synthesis of enzymes of the galactose operon of Escherichia coli

Summary Polar mutations of the galactose operon of both, nonsense and insertion type have been studied in a system for DNA-dependent synthesis of the galactose enzymes of Escherichia coli. In vivo, these mutations reduce to different degrees the level of expression of the gene located on the promoter-distal side of the mutation. No such polar effects are observed in vitro. This relief of polarity is neither due to the action of nonsense suppressors, nor to random initiation of mRNA synthesis.A special aspect of this study concerns those insertion mutations which carry a segment of DNA of foreign origin inserted near the control region of the galactose operon. In vivo, mutants of this type produce only one percent or less of the three galactose enzymes as compared to the wildtype. The residual enzyme synthesis is not or only slightly affected by inducer. In contrast, DNA carrying such insertion mutations is fully active in the cell-free enzyme synthesis and sensitive to the controls exerted by the galactose repressor and by the catabolite gene activator protein.     

DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Summary Two active enzymes of the galactose operon of Escherichia coli, uridyl transferase and galactokinase have been synthesized with high yields in a DNA dependent system for protein synthesis. The unspecific blank values amount to less than two percent of the rate obtained under optimal conditions and permit the accurate determination of even a small fraction of the maximum synthesis rate. Therefore this system provides a sensitive assay for the biological activity of DNA that contains the intact galactose operon of Escherichia coli.The synthesis of these galactose enzymes is to a high extent dependent on the presence of cyclic adenosine-3:5-monophosphate.D-fucose, known as an inducer of the galactose operon in vivo, stimulates the synthesis of galactokinase, indicating that the repressor of the galactose operon in active under these conditions. This stimulation is not observed, if the bacterial extract is prepared from a strain defective for the galactose repressor or if the DNA carries an operator constitutive mutation in the galactose operon. Therefore the stimulation by D-fucose is true derepression.     

Overexpression and purification of the galactose operon enzymes from Escherichia coli

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically characterized by gel-filtration column chromatography and isoelectric focusing, and the Km values for their substrates were determined.     

Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis

Escherichia coli extracts activate cell-free protein synthesis systems by providing the catalysts for translation and other supporting reactions. Recent results suggest that high-density fermentations can be used to provide the source cells, but the subsequent cell extract preparation procedure requires multiple centrifugation and dialysis steps as well as an expensive runoff reaction. In the work reported here, the extract preparation protocol duration was reduced by nearly 50% by significantly shortening several steps. In addition, by optimizing the runoff incubation, overall reagent costs were reduced by 70%. Nonetheless, extracts produced from the shorter, less expensive procedure were equally active. Crucial steps were further examined to indicate minimal ribosome loss during the standard 30,000g centrifugations. Furthermore, sucrose density centrifugation analysis indicated that although an incubation step significantly activates the extract, ribosome/polysome dissociation is not required. These insights suggest that consistent cell extract can be produced more quickly and with considerably less expense for large-scale cell-free protein production, especially when combined with high-density fermentation protocols.     

Effect of rate-limiting elongation on bacteriophage MS2 RNA-directed protein synthesis in extracts of Escherichia coli

The consequences of limiting the rate of elongation of protein synthesis in vitro have been examined. The concentration of Trp-tRNA^Trp was manipulated by varying the amount of exogenously added tryptophan in extracts from an Escherichia coli mutant in which the tryptophanyl-tRNA-synthetase has a higher K_M for tryptophan. The evidence presented supports the hypothesis that variation of the rate of elongation can be a means of regulating gene expression, both directly, by slowing or accelerating the rate of protein synthesis and indirectly, by leading to varying three-dimensional structures of the messenger RNA when progress of the ribosomes is perturbed. The data can be described by assuming that if a specific transfer RNA is limiting, to a first approximation the overall rate of protein synthesis is determined by the relative rate of reading past an individual codon requiring that tRNA raised to the power of how many times that codon appears in the message. This could be explained by a model in which, with a significant probability, the ribosome stops protein synthesis prematurely at these codons, falls off the messenger RNA and is available for further rounds of protein synthesis. In agreement with other work, evidence is also presented that suggests that under the most drastic available limitation of the elongation rate, that is, starvation for a given amino acid, reading through the corresponding “hungry codon” occurs in vitro at a surprisingly high rate, possibly due to mistranslation.     

Sequential appearance of the galactose enzymes in E. coli

Summary Upon induction, the three galactose enzymes appear in the order epimerase, transferase, kinase. Introduction of an amber mutation into the transferase gene seems to shorten the time interval between addition of inducer and the appearance of kinase. The time of appearance of epimerase is not influenced by amber mutations in the transferase gene.     

Effect of zeolites on protein synthesis in a cell-free system from Escherichia coli

Summary The effects of zeolites on protein synthesis in a cell-free system were investigated. The efficiency of protein synthesis was markedly enhanced upon the addition of zeolites to the reaction mixture. Pretreatment of reaction mixture with the zeolite prior to the start of reaction also stimulated the protein synthesis indicating that the effect is at least partially due to the removal of inhibitory substance(s) from the reaction mixture.     

An easy cell-free protein synthesis system dependent on the addition of crude Escherichia coli tRNA

The protein-synthesizing S30 extract of Escherichia coli contains tRNA, which limits its applications in cell-free protein synthesis. Here, we show that at least Arg- and Ser-acceptor activities can be removed from a standard S30 extract by treatment with an immobilized RNase A resin. This RNase-treated extract exhibits no protein synthesis activity, but regains it when supplied with crude E. coli tRNA and a small amount of human placental RNase inhibitor. The protein synthesis is dependent on the addition of tRNA in the presence of the RNase inhibitor. Chloramphenicol acetyltransferase was synthesized with this system and found to be active.     

Effects of Escherichia coli ribosomal protein S12 mutations on cell-free protein synthesis.

We examined the effects of Escherichia coli ribosomal protein S12 mutations on the efficiency of cell-free protein synthesis. By screening 150 spontaneous streptomycin-resistant isolates from E. coli BL21, we successfully obtained seven mutants of the S12 protein, including two streptomycin-dependent mutants. The mutations occurred at Lys42, Lys87, Pro90 and Gly91 of the 30S ribosomal protein S12. We prepared S30 extracts from mutant cells harvested in the mid-log phase. Their protein synthesis activities were compared by measuring the yields of the active chloramphenicol acetyltransferase. Higher protein production (1.3-fold) than the wild-type was observed with the mutant that replaced Lys42 with Thr (K42T). The K42R, K42N, and K42I strains showed lower activities, while the other mutant strains with Lys87, Pro90 and Pro91 did not show any significant difference from the wild-type. We also assessed the frequency of Leu misincorporation in poly(U)-dependent poly(Phe) synthesis. In this assay system, almost all mutants showed higher accuracy and lower activity than the wild-type. However, K42T offered higher activity, in addition to high accuracy. Furthermore, when 14 mouse cDNA sequences were used as test templates, the protein yields of nine templates in the K42T system were 1.2-2 times higher than that of the wild-type.     

Strains of Escherichia coli with simultaneous elevated synthesis of three catabolic enzymes

Summary Escherichia coli strains producing simultaneously three enzymes (tryptophanase, D-serine deaminase and -galactosidase) in elevated amounts, and with increased resistance to catabolite repression were isolated by a multistep chemostat selection procedure.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system