Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli.
The ATP-dependent Clp protease of Escherichia coli contains two dissimilar components: the Clp A regulatory polypeptide, with two ATP binding sites and intrinsic ATPase activity, and the Clp P subunit, which contains the proteolytic active site. The DNA sequence of the clpP gene predicts a protein of 207 amino acids (Mr 21,679), which is in close agreement with the size determined by sodium dodecyl sulfate-gel electrophoresis of purified Clp P. Clp P has a native Mr of approximately 240,000, and electron micrographs of the protein show superimposed disk-like structures with a central cavity, similar in appearance to purified proteasomes from eukaryotic cells. Clp P is synthesized with a 14-amino acid leader which is rapidly cleaved in vivo to yield the 193-amino acid protein which has activity in vitro. The clpP gene is at 10 min on the E. coli map, close to that for the ATP-dependent Lon protease of E. coli and far from the gene for clpA. Primer extension experiments indicate that transcription initiates immediately upstream of the coding region for Clp P, with a major transcription start at 120 bases in front of the start of translation. Insertion mutations in clpP have been isolated and transferred to the chromosome; strains devoid of Clp P are viable in the presence or absence of Lon protease. Mutations in clpP stabilize the same Clp A-beta-galactosidase fusion protein specifically stabilized by clpA mutations, providing the first genetic evidence that Clp A and Clp P act together in vivo.
Laboratory of Molecular Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
M R
MR
Maurizi
W P
WP
Clark
Y
Y
Katayama
S
S
Rudikoff
J
J
Pumphrey
B
B
Bowers
S
S
Gottesman
D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D049070:Protease La; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
D000596:Amino Acids; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011498:Protein Precursors; D012333:RNA, Messenger; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La