A. Translational Efficiency of the Adenylate Cyclase Gene. Since the analysis of the structure of the adenylate cyclase gene revealed that the initiation codon was UUG rather than the usual AUG, it was suggested that this feature of the gene structure might play a regulatory role. We tested the possibility that the UUG codon decreases the efficiency of translation. By the use of recombinant DNA procedures, we constructed plasmids containing the gene for adenylate cyclase in which the initiation codon was UUG, GUG or AUG. Tests of the expression of adenylate cyclase activity by these plasmids revealed that the AUG initiation codon promoted from three to five times as much gene expression as did the UUG initiation codon. These results support the hypothesis that the UUG initiation codon limits the expression of the adenylate cyclase gene. B. Reconstitution of Regulatory Properties of Adenylate Cyclase in Escherichia coli Extracts. Based on studies carried out using intact or premeabilized cells, it has been proposed that the regulatory properties of E. coli adenylate cyclase require an interaction of this enzyme with proteins of a unique multienzyme sugar transport system. In addition, evidence has been presented that a functional interaction is only observed in the presence of inorganic phosphate. These ideas were put on a firmer basis by a reconstitution approach, using a purified preparation of E. coli adenylate cyclase and homogeneous preparations of the transport proteins. In these experiments, we were successful in reconstituting all the regulatory properties of adenylate cyclase observed in intact cell preparations.
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