PMID: 7025892Jul 7, 1981Paper

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

Biochemistry
M I PoulisI G Young

Abstract

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. Bio...Continue Reading

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Citations

Jul 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·J BouvierP Stragier
Sep 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·P ReddyK McKenney
Oct 15, 1984·European Journal of Biochemistry·H D CampbellI G Young
Jul 1, 1985·European Journal of Biochemistry·G WeyensA Piérard
Nov 21, 1983·Journal of Theoretical Biology·R Mikelsaar
Oct 8, 1999·Archives of Biochemistry and Biophysics·V A RapisardaE M Massa
Apr 19, 1996·The Journal of Biological Chemistry·R W MorganJ E Donelson
Mar 24, 2017·Nucleic Acids Research·Ariel HechtMarc Salit
Sep 1, 1984·Microbiological Reviews·W J Ingledew, R K Poole
Oct 25, 1985·Science·W T Wickner, H F Lodish

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