Site-directed mutagenesis of the dual translational initiation sites of the clpB gene of Escherichia coli and characterization of its gene products.
The heat shock protein ClpB in Escherichia coli is a protein-activated ATPase and consists of two proteins with sizes of 93 and 79 kDa. By polymerase chain reaction-aided site-directed mutagenesis, both the proteins have been shown to be encoded by the same reading frame of the clpB gene, the 93-kDa protein (ClpB93) from the 5'-end AUG translational initiation site and the 79-kDa protein (ClpB79) from the 149th codon (an internal GUG start site). Both the purified ClpB93 and ClpB79 proteins behave as tetrameric complexes with a very similar size of about 350 kDa upon gel filtration on a Superose-6 column. Both appear to be exclusively localized to the cytosol of E. coli. Both show inherent ATPase activities and have an identical Km of 1.1 mM for ATP. The ATPase activity of ClpB93 is as markedly stimulated by proteins, including casein and insulin, as that of wild-type ClpB, but the same proteins show little or no effect on ClpB79. Because ClpB79 lacks the 148 N-terminal sequence of ClpB93 but retains the two consensus sequences for adenine nucleotide binding, the N-terminal portion appears to contain a site(s) or domain(s) responsible for protein binding. Furthermore, ClpB79 is capable of inhibiting the casein-activated ATPase activity of ClpB93 in a dose-dependent manner but without any effect on its inherent ATPase activity. In addition, ClpB93 mixed with differing amounts of ClpB79 behave as tetrameric molecules, although its protein-activated ATPase activity is gradually reduced. These results suggest that tetramer formation between ClpB93 and ClpB79 may be responsible for the inhibition of the activity.
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
S K
SK
Park
K I
KI
Kim
K M
KM
Woo
J H
JH
Seol
K
K
Tanaka
A
A
Ichihara
D B
DB
Ha
C H
CH
Chung
D000251:Adenosine Triphosphatases / Q000096:biosynthesis*; D001483:Base Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis; D015183:Restriction Mapping
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D009838:Oligodeoxyribonucleotides; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C069775:ClpB protein, E coli