Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants.
Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the alpha and beta subunits of luciferase from Vibrio harveyi, such that luciferases with normal alpha subunits and variant beta subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T.O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., & Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3' region of luxB were divided into three groups: (A) those with deletions in the 3' untranslated region that left the coding sequences intact, (B) those that left the 3' untranslated sequences intact but deleted short stretches of the 3' coding region of the beta subunit, and (C) those for which the 3' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3' to the translational open reading frame of the beta subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact.
Sugihara
J
J
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843.
Baldwin
T O
TO
D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D004262:DNA Restriction Enzymes; D005796:Genes; D005798:Genes, Bacterial; D008156:Luciferases / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013696:Temperature; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D046911:Macromolecular Substances; D008156:Luciferases; D004262:DNA Restriction Enzymes