Preincubated S30 system

ZuBAY and coworkers (Zubay and Chambers, 1969) have established a modification of the S30 system of Matthaei and Nirenberg (I96I), a preincubated S30 system. This system found wide application for the study of synthesis of E, coli enzymes. The S30 is mixed with a small volume of a solution containing all the components needed for translation (amino acids, ATP, ATP-regenerating system) and preincubated at 37° for 80 minutes. The S30 extract is then dialyzed against a desired buffer at 0°. Table 2 summarized the preparation of the S30 system and lists the constituents of the incubation mixture. 

In addition to magnesium, the concentration of calcium salts appears to be critical in the preincubated S30 system (Table 2 Zubay, personal communication). 

How does the rate of in vitro enzyme synthesis compare to in vivo synthesis Under optimal conditions, 0.2 [Jig / -galactosidase per ml is synthesized within 60 minutes of incubation (Chambers and Manley, 1973). Compared to maximal in vivo synthesis, this corresponds to an efficiency of 0.06%. In 08Odarg directed synthesis of N-a-acetyl-L-ornithinase, the in vitro rate was approximately 0.09% of the in vivo rate (Urm et al., 1973). T7 infected cells produce about 7X10 units of lysozyme per minute and per mg of ribosomes assuming that 3 X 10 cells contain 1 mg (1 500 O.D.geo) of ribosomes. The in vitro rate is 4 X 10 units per minute and per mg of ribosomes or 0.57% of the in vivo rate. All these data are of course approximations. Rates of enzyme synthesis in both the preincubated S30 system and the DEAE system are of the same order of magnitude. 

Tables I and II catalog the wide variety of mRNA fractions which have been faithfully translated in the rabbit reticulocyte lysate and the preincubated S30 of Krebs II cells without the addition of homologous tissue fractions. In addition, the mRNA s for mouse and rabbit globin have been translated in the preincubated mouse (and rat) liver system (Sampson et al., 1972 Sampson and Borghetti, 1972). Rabbit globin, immunoglobulin light chain, and collagen mRNA s have been translated in the Xenopus eggs and oocytes (see Chapter 4, this volume). The most obvious interpretation of these results is that messenger-specific initiation factors do not exist in animal cells and that there is no restriction on the range of mRNA s that a tissue can translate.

In a crude extract or S30 system, no additional tRNA is required. The S30 or crude extract contains saturating amounts of tRNA. The DEAE-system is partially dependent on the addition of tRNA the protein fraction is almost free of tRNA, but tRNA is attached to the ribosomes. In fact, preincubated ribosomes still contain sufficient tRNA to allow 30-50% of the leucine incorporation which can be achieved upon addition of tRNA. However, some tRNA species are apparently limiting since meaningful protein synthesis is reduced to 5% (Gold and Schweiger, 1969a). Response to tRNA addition is improved by further purification of the ribosomes. 

Transcription of natural templates like T4 and TZ DNA by E, coli RNA polymerase yields defined RNA products at both high and low ionic strength and without the help of additional factors (Millette et al., 1970). At the ionic strength of the in vitro protein synthesizing systems (preincubated S30 or... 

In an S30 extract from thermophilic bacteria, the endogenous DNA can be efficiently digested by incubation with pancreatic DNase at low temperature (G. Bauer, W. Siegert and P. H. Hofschneider, personal communication). The DNase is then destroyed by preincubation at the growth temperature of the bacteria. The system has been used for the synthesis of coat proteins under the direction of 0(x-4 phage DNA (Rabussay et al., I969), the products being detected by gel electrophoresis and immunoprecipitation (Bauer et al., personal communication). 

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