PMID:3042779
| Citation |
Chin, DT, Goff, SA, Webster, T, Smith, T and Goldberg, AL (1988) Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La. J. Biol. Chem. 263:11718-28 |
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| Abstract |
To learn more about the mechanism and regulation of the ATP-dependent protease La in Escherichia coli, the lon gene was completely sequenced using the dideoxy method on fragments generated by Bal31 digestion. The predicted amino acid composition based on the DNA sequence agreed well with the composition of the acid-hydrolyzed protease. The predicted NH2-terminal amino acid sequence, tryptophan content, and the carboxyl terminus also agreed with experimental data. However, the molecular weight of 87,000 (783 amino acids) calculated from the DNA sequence was lower than prior estimates. The tetrameric enzyme contains four binding sites for ATP, a DNA-binding domain, a proteolytic site, and a regulatory site that binds unfolded polypeptides. An ATP-binding pocket exists on each subunit as shown by consensus sequences and elements of secondary structure resembling those on other nucleotide-binding proteins (e.g. adenylate kinase, RecA). For this purpose, improved consensus patterns for identifying ATP-binding domains were developed. Computer-assisted comparisons, however, failed to demonstrate any regions homologous to sequences in other polypeptides including proteases or DNA-binding proteins. This enzyme also contains an unusual highly acidic domain surrounded by very basic sequences. Protease La is the first ATP-dependent protease sequenced and seems to represent a new type of enzyme. The promoter sequence was similar to consensus sequences for other heat-shock promoters. Using site-directed mutagenesis, alterations were introduced into the putative promoter sequence. Mutations upstream of -35 had little effect, but alterations immediately upstream of -10 lowered basal transcription of a lon-lacZ operon fusion and reduced its response to inducers of the heat-shock response. |
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| Keywords |
ATP-Dependent Proteases; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Base Sequence; Binding Sites; DNA/metabolism; DNA, Bacterial/genetics; DNA, Recombinant; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Genes, Bacterial; Heat-Shock Proteins; Hot Temperature; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Mutation; Plasmids; Promoter Regions, Genetic; Protease La; Sequence Homology, Nucleic Acid; Serine Endopeptidases/genetics |
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