The Isolation and Structure of Operator
After the isolation of lac repressor and the demonstration that it bound to DNA, interest turned to the structure of operator. The first questions concerned its size and sequence. Answering such questions is now straightforward with the application of genetic engineering techniques. The questions about the lac operator, however, came to the fore before the era of genetic engineering. Because ingenious techniques were developed and the work directly led to the development of many of the genetic engineering techniques now used, we will review the techniques, now outdated, used to isolate and sequence the lac operator.
Before developing their chemical sequencing method, Gilbert and Maxam sequenced the operator, primarily by direct DNA sequencing methods, whereas somewhat later Reznikoff, Barnes, Abelson, and co-workers sequenced the entire lac regulatory region by purifying and
Figure 11.9 The DNA sequence of thelacregulatory region and the bindingsites of the proteins that bind there.
sequencing RNA copies of it (Fig. 11.9). Gilbert’s method required isolation of pure operator itself. DNA was isolated from phage carrying the lac genes. This DNA was sonicated to fragments about 1,000 base pairs long, repressor was added, and the mixture was passed through a cellulose nitrate filter. The DNA fragments containing operator were bound by repressor and held on the filter whereas the rest passed through. Then the operator-containing fragments were specifically re-leased by adding IPTG to the rinse buffer. Finally, repressor was again bound to these fragments, and the portions of the fragments not pro-tected by repressor were digested by DNAse. The operator was found to be about 20 base pairs long, and it had been purified 2000-fold from the phage or 20,000-fold from the E. coli DNA!
The lac operator possesses a symmetrical sequence. This suggested that two of the repressor subunits symmetrically made contact with it (Fig. 11.10). To probe the structure of lac-operator-repressor complexes, Gilbert and Maxam turned to DNA protection studies. They bound repressor to operator and investigated the reaction of dimethylsulfate
Figure 11.10 The symmetry inlacoperator and how a protein consisting oftwo identical subunits could bind to it.
with bases of the operator in the complex. Indeed, a symmetrical protection pattern was obtained, but, more important, they noticed that the method could easily and rapidly be adapted to yield the actual sequence of a DNA fragment. From this beginning they developed the chemical method for sequencing DNA.
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