Detection and Isolation of AraC Protein
However ingenious, genetic and physiological or cloning and mapping experiments suffer from the defect that they are rarely able to provide rigorous proofs of mechanisms of action. Proof of a model usually requires purification of the system’s individual components and in vitro reconstruction of the system. How then could biologically active AraC protein be purified for biochemical studies? Detection of the lac re-pressor, which had been accomplished earlier, was difficult enough. Its isolation capitalized upon its tight-binding to an inducer of the lac operon, IPTG. Not even this handle was available for detection of AraC protein. In vivo experiments measuring the induction level of arabinose enzymes in cells as a function of the intracellular arabinose concentra-tion showed that the affinity of AraC protein for arabinose was too low to permit its detection by the equilibrium dialysis that was used to isolate lac repressor.
Work on the isolation of AraC was performed well before genetic engineering permitted facile isolation of many proteins. Now proteins often can be detected and purified merely by
engineering such a great hypersynthesis that cell lysates possess a prominent
new band upon SDS gel electrophoresis. Such gels then provide a way to follow
the purifica-tion of the overproduced protein, but they do not ensure that the
protein is biologically active. How can an assay be devised for the biological
activity of AraC protein? The only activity that possesses sufficient
sensitivity was based on the ability of AraC protein to induce synthesis of the
araBAD genes. Working with the lac operon, Zubay had developed a means
of preparing a partially fractionated cell lysate in which added DNA could be
transcribed and translated. When a concentrated source of lacZ DNA is added to this system, lac mRNA is synthesized and translated into active β-galactosidase. This system was adapted to the
arabinose genes to search for the AraC protein.
Figure 12.6 Schematic of the coupled transcription-translation system.
The necessary coupled transcription-translation system consists of a cell extract from which much cellular DNA and all mRNA has been removed (Fig. 12.6). Then DNA template, salts, amino acids, and en-zyme cofactors are added. The cell extracts must be made from araB mutants so that the small quantities of the araB enzyme, ribulokinase, which will be synthesized in vitro, can be detected. Also, the extracts must be free of AraC protein so that it can be detected on its addition to the extracts. Of course, it is sensible that the source of AraC protein be as concentrated as possible as well as lack ribulokinase. Finally, a highly concentrated source of araB DNA must be added to the synthesis extract.
It was possible to meet the various requirements of the in vitro system. The extract for in vitro synthesis was prepared from cells deleted for the araCBAD genes. The source of AraC protein was the araCBAD deleted cells infected with a phage carrying the araC gene but not an active araB gene. The source of araB DNA was a phage carrying the arabinose genes. Of course, similar experiments done at the present time would utilize plasmids carrying the desired genes. When the ingredients were mixed, AraB protein was synthesized only if both arabinose and AraC-containing extract were added to the reaction. This result rigorously excludes the possibility that AraC protein only appears to regulate positively because its actual role is transporting arabinose into cells. It also makes the possibility of a double repressor regulation system exceedingly remote.
Of course, the last word on the positive nature of the regulation system came with a completely pure in vitro transcription system consisting of just cyclic AMP receptor protein, RNA polymerase, ara DNA, and AraC protein plus arabinose, cAMP and triphosphates. Ara-specific messenger is synthesized if and only if these components and arabinose are present in the reaction. The fact that this system works also shows that the regulation is exerted at the level of initiation of transcription rather than via degradation of mRNA or even at a trans-lational level.
Although the assay was cumbersome, the coupled transcription-translation system permitted quantitation of AraC protein in fractions obtained from purification steps. Micrograms of the protein could be purified. When the araC gene was fused to the lacZ promoter and placed on a high-copy-number plasmid the level of AraC protein reached values permitting straightforward purification. Now it is possible in just a few days to purify 20 milligrams of the protein. This facilitates physical experiments designed to examine the basis of the protein’s activities.
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