In vitro synthesis of the respiratory NADH dehydrogenase of Escherichia coli. Role of UUG as initiation codon.
The respiratory NADH dehydrogenase of Escherichia coli has been synthesized in vitro in a coupled transcription--translation system with cloned deoxyribonucleic acid (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 were shown to comigrate with authentic methionine-containing tryptic peptides from the purified enzyme. Using a transcription-translation system derived from an ndh mutant, it was shown 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 three different species, with N-formylmethionine, methionine, or threonine at the N terminus. The results indicated that only partial N-terminal processing was occurring in vitro and that the first residue of the unprocessed NADH dehydrogenase is N-formylmethionine. Since DNA sequencing has shown that this residue is encoded by UUG [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. (in press)], this work verifies the role of UUG as a normal initiation codon.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics*; D003579:Cytochrome Reductases / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D014158:Transcription, Genetic
D000596:Amino Acids; D003062:Codon; D004269:DNA, Bacterial; D012333:RNA, Messenger; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase