DNA-DIRECTED PEPTIDE SYNTHESIS, I. THE SYNTHESIS OF THE a-FRAGMENT OF THE ENZYME f-GALACTOSIDASE*

The central working hypothesis of molecular genetics states that DNA directs the synthesis of an RNA which in turn directs the synthesis of protein. There can be little doubt as to the validity of this two-step process; nonetheless, its utmost usefulness will only be realized when it has been fully duplicated in cell-free systems. The second step of this process, RNA-directed protein synthesis, has been carried out in cell-free systems. For example, specific viral messenger RNA has been purified and characterized, added to a cell-free extract prepared from E. coli, and therein stimulated the de novo synthesis of coliphage coat protein. ' The first step of the process, DNA-directed RNA synthesis, has been demonstrated only in part; various investigators have shown that DNA primes the synthesis of an RNA which is complementary to part of its sequence. It remains to be shown that RNA made on a DNA template in vitro can be used to make the same polypeptide chain that it would make in the cell. It is with this goal that we have attempted to find suitable conditions for DNA-directed enzyme synthesis. We believe our results have been sufficiently successful to warrant a brief report. For various reasons the system chosen for study was the enzyme ,3-galactosidase of E. coli. Previous attempts have been made to synthesize this enzyme, but the incorporating systems were comparatively poor and the evidence lacking in rigor so that those attempts can be discounted. f3-Galactosidase has a molecular weight of 535,000 and is known to be a tetramer containing identical subunits.2 It can be calculated that the monomer contains 1170 amino acid residues.3 Since the monomer is so large, it may be difficult to synthesize detectable amounts of complete molecules in vitro; therefore systems were sought in which only a fragment of this long polypeptide chain could be detected. Two such assay systems exist which utilize the process of intracistronic complementation. The polypeptide chain has been divided into parts designated a, f3, and w.4, 5 (Synthesis begins from the a end.) Both a and w complementation have been demonstrated.4 5 In co complementation, for example, if one mixes a cell extract from an acceptor strain which has a mutation in the X region with an extract from a donor strain which produces a normal w region, or indeed with isolated w peptides, a high level of f3-galactosidase activity is obtained.5 Alpha complementation is analogous and is best for our purpose because we need synthesize only the a-fragment, complement with a mutant which has a deletion of the a region, and assay for 13-galactosidase activity. The DNA used in these studies is of critical importance. The DNA used was not from the E. coli chromosome but from the virus 480 dlac which carried the fl-galactosidase gene in a concentration of about 60 times greater than present in E. coli. This DNA was prepared from a doubly lysogenic strain of E. coli and was subjected to rigorous purificationl by a procedure described elsewhere.6 In order to ensure that any f3-galactosidase arising in the incorporation system is the result of DNA-directed, de novo synthesis, the E. coli cells used for preparing the