Partial loss of function mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, affect highly conserved amino acids implicated in ATP binding and hydrolysis.
A set of 37 mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, was isolated using a selection for high constitutive expression of heat shock proteins. Of these, 11 mutants were able to carry out some but not all functions of DnaK. These partial function mutants were divided into two classes. Class I mutants are recessive and permit replication of bacteriophage lambda and growth of cells up to 40 degrees C. Class II mutants are dominant, do not permit growth of lambda, and are temperature-sensitive for growth above 34 degrees C. Mutations in both classes alter amino acids that are highly conserved in the 70-kDa heat shock protein family. The dominant negative mutations provide strong genetic evidence that at least one form of DnaK is multimeric. Moreover, every dominant negative mutation occurs at an amino acid that has been hypothesized to be intimately involved in the process of ATP binding and hydrolysis. Our findings provide strong support for the hypothesis that such mutations are excellent tools for identifying amino acids that play critical roles in protein function.
Wild
J
J
Department of Bacteriology, University of Wisconsin-Madison 53706.
Kamath-Loeb
A
A
Ziegelhoffer
E
E
Lonetto
M
M
Kawasaki
Y
Y
Gross
C A
CA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda / Q000502:physiology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005799:Genes, Dominant; D005804:Genes, Lethal; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009154:Mutation; D010641:Phenotype; D013696:Temperature; D014779:Virus Replication
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007544:Isopropyl Thiogalactoside; D015500:Chloramphenicol O-Acetyltransferase; C063842:dnaK protein, E coli