In vitro synthesis of the respiratory dehydrogenase of Escherichia coli. Role of UUG as initiation codon

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Abstract
The respiratory NADH dehydrogenase of E. coli was synthesized in vitro in a coupled transcription-translation system with cloned DNA as template. The identity of the protein produced was confirmed by paper chromatography and electrophoresis of tryptic peptides. [35S]Methionine-labeled tryptic peptides from the in vitro product comigrated with authentic methionine-containing tryptic peptides from the purified enzyme. A transcription-translation system derived from an ndh mutant was used to show that the enzyme produced in vitro was incorporated into membrane vesicles of the mutant to give functional, cyanide-sensitive NADH oxidase activity. Radiochemical N-terminal sequencing of the synthesized NADH dehydrogenase showed that the product was a mixture of 3 different species, with N-formylmethionine, methionine or threonine at the N terminus. Only partial N-terminal processing occurred in vitro and the 1st residue of the unprocessed NADH dehydrogenase is N-formylmethionine. Since DNA sequencing has shown that this residue is encoded by UUG (Young I. G. et al., 1981) the role of UUG as a normal initiation codon is verified.