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 p olymerase chain reaction-aided site-directed mutagenesis, both the pro teins have been shown to be encoded by the same reading frame of the c lpB gene, the 93-kDa protein (ClpB93) from the 5'-end AUG translationa l initiation site and the 79-kDa protein (ClpB79) from the 149th codon (an internal GUG start site). Both the purified ClpB93 and ClpB79 pro teins 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 A TPase activities and have an identical K(m) of 1.1 mM for ATP. The ATP ase activity of ClpB93 is as markedly stimulated by proteins, includin g 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-te rminal 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, C lpB79 is capable of inhibiting the casein-activated ATPase activity of ClpB93 in a dose-dependent manner but without any effect on its inher ent ATPase activity. In addition, ClpB93 mixed with differing amounts of ClpB79 behave as tetrameric molecules, although its protein-activat ed ATPase activity is gradually reduced. These results suggest that te tramer formation between ClpB93 and ClpB79 may be responsible for the inhibition of the activity.