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

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Abstract
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.