A distinct segment of the sigma 32 polypeptide is involved in DnaK-mediated negative control of the heat shock response in Escherichia coli.
Induction of heat shock proteins in Escherichia coli is caused by a transient increase in the cellular level of sigma 32 (the rpoH gene product), a protein required for transcription of heat shock genes. Both increased synthesis and stabilization of sigma 32 contribute to the increase in sigma 32. We previously showed that heat-induced translation of sigma 32-beta-galactosidase fusion protein encoded by an rpoH-lacZ gene fusion was mediated by an mRNA secondary structure formed between two 5'-proximal segments (A and B) of rpoH coding sequence spanning some 200 nt. We now report that a portion of the sigma 32 polypeptide that corresponds to further downstream (designated region C) is involved in the DnaK-mediated negative control resulting in the shutoff of heat-induced synthesis and degradation of fusion protein. Gene fusions carrying the 5' half (433 nt) or more of the rpoH coding sequence exhibited normal shutoff of synthesis, and the fusion proteins produced were very unstable, like authentic sigma 32; both the shutoff of synthesis and the instability of protein were markedly affected by the dnaK and dnaJ mutations. In contrast, gene fusions carrying < or = 364 nt (lacking region C) and a fusion carrying most of the rpoH sequence but with a frameshift mutation specifically affecting region C exhibited little or no shutoff and produced stable proteins. These results indicate that a distinct segment of sigma 32 plays a critical role in the negative feedback control of sigma 32. The control may be exerted during or after completion of sigma 32 synthesis mediated by interaction between nascent or mature sigma 32 and DnaK/DnaJ proteins.
Nagai
H
H
Institute for Virus Research, Kyoto University, Japan.
Yuzawa
H
H
Kanemori
M
M
Yura
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D016368:Frameshift Mutation; D005798:Genes, Bacterial; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis; D006358:Hot Temperature; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D012808:Sigma Factor / Q000096:biosynthesis / Q000378:metabolism*; D014157:Transcription Factors; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; D008715:Methionine; D001616:beta-Galactosidase; C063842:dnaK protein, E coli