The C Terminus of ς(32) Is Not Essential for Degradation by FtsH

A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific ς(32) subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including ς(32), as degradation signals for AAA proteases. We investigated the role of the C terminus of ς(32) in FtsH-dependent degradation by analysis of C-terminally truncated ς(32) mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of ς(32) did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of ς(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated ς(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of ς(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.