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On the mechanism of FtsH‐dependent degradation of the σ 32 transcriptional regulator of Escherichia coli and the role of the DnaK chaperone machine
Author(s) -
Blaszczak Adam,
Georgopoulos Costa,
Liberek Krzysztof
Publication year - 1999
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1999.01155.x
Subject(s) - biology , chaperone (clinical) , proteolysis , escherichia coli , rna polymerase , protease , mutant , protein degradation , biochemistry , rna , proteases , microbiology and biotechnology , enzyme , gene , medicine , pathology
The Escherichia coli σ 32 transcriptional regulator has been shown to be degraded both in vivo and in vitro by the FtsH (HflB) protease, a member of the AAA protein family. In our attempts to study this process in detail, we found that two σ 32 mutants lacking 15–20 C‐terminal amino acids had substantially increased half‐lives in vivo or in vitro , compared with wild‐type σ 32 . A truncated version of σ 32 , σ 32 CΔ, was purified to homogeneity and shown to be resistant to FtsH‐dependent degradation in vitro , suggesting that FtsH initiates σ 32 degradation from its extreme C‐terminal region. Purified σ 32 CΔ interacted with the DnaK and DnaJ chaperone proteins in a fashion similar to that of wild‐type σ 32 . However, in contrast to wild‐type σ 32 , σ 32 CΔ was largely deficient in its in vivo and in vitro interaction with core RNA polymerase. As a consequence, the truncated σ 32 protein was completely non‐functional in vivo , even when overproduced. Furthermore, it is shown that wild‐type σ 32 is protected from degradation by FtsH when complexed to the RNA polymerase core, but sensitive to proteolysis when in complex with the DnaK chaperone machine. Our results are in agreement with the proposal that the capacity of the DnaK chaperone machine to autoregulate its own synthesis negatively is simply the result of its ability to sequester σ 32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease.