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 less than or equal to 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.