ENZYMES OF THE LACTOSE OPERON IN E. COLI

tinues coordinately with the other enzymes of the operon. When the inducer is removed (deinduction), their synthesis rapidly stops. It has been proposed' that the inducer inmitiates the synthesis of the messenger RNA corresponding to the genes of the operon. On deinduction, further messenger synthesis ceases, and those molecules already present are rapidly degraded. Although less well established, it is also thought that an individual messenger molecule contains the information for the synthesis of all the enzymes of the corresponding operon."2- Furthermore, it has been suggested that on each messenger molecule there is only a single point for the initial attachment of ribosomes, each of which then translate the cistrons of the operon in the order of their linear representationi on the messenger.' Studies on the early kinetics of 3-galactosidase induction' 7 have shown that the lag between the addition of inducer and the time at which the rate of enzyme synthesis becomes constant is the order of 2-3 min at 37?. When the inducer is removed, the lag before galactosidase synthesis stops is of almost equal duration., 7 These lags have generally been interpreted in terms of the rates of messenger synthesis and decay, respectively.8 ' In the present communication, using a recently developed sensitive spectrophotometric assay for thiogalactoside transacetylase, 10 we compare the early kinetics of induction and deinduction of this enzyme with those of f-galactosidase. We find that on initiation of induction, transacetylase synthesis begins 2-3 min later than that of 3-galactosidase and, on deinduction, the synthesis of the transacetylase continues for about 2 min after galactosidase formation has stopped. The evidence presently availablet suggests that the galactosidase gene lies closer to the operator than the gene-controlling transacetylase synthesis. Therefore, the findings detailed herein indicate that the lac messenger is translated sequentially starting from the operator end. It also appears that, on deinduction, the messenger is sequentially degraded in the same direction. Materials and Methods.-Isopropyl 0-d-thiogalactoside (IPTG) and o-nitrophenyl galactoside (ONPG) were purchased from Mann Research Laboratories and dithionitrobenzoic acid from the Aldrich Chemical Co. The strains of E. coli used in the present study are listed in Table 1. Bacteria were grown at 37? with vigorous aeration in a New Brunswick gyrotory shaker. The synthetic medium used was 0.5% in glycerol and contained 0.5 mg/ml thiamine hydrochloride and casamino acids as described.1? Under these conditions the doubling time of the cells was about 80 min.