In E. coli, protein degradation plays important roles in regulating the levels of specific proteins and in eliminating damaged or abnormal proteins. E. coli possess a very large number of proteolytic enzymes distributed in the cytoplasm, the inner membrane, and the periplasm, but, with few exceptions, the physiological functions of these proteases are not known. More than 90% of the protein degradation occurring in the cytoplasm is energy-dependent, but the activities of most E. coli proteases in vitro are not energy-dependent. Two ATP-dependent proteases, Lon and Clp, are responsible for 70-80% of the energy-dependent degradation of proteins in vivo. In vitro studies with Lon and Clp indicate that both proteases directly interact with substrates for degradation. ATP functions as an allosteric effector promoting an active conformation of the proteases, and ATP hydrolysis is required for rapid catalytic turnover of peptide bond cleavage in proteins. Lon and Clp show virtually no homology at the amino acid level, and thus it appears that at least two families of ATP-dependent proteases have evolved independently.

Proteases and protein degradation in Escherichia coli.

Mu transposition is promoted by an extremely stable complex containing a tetramer of the transposase (MuA) bound to the recombining DNA. Here we purify the Escherichia coli ClpX protein, a member of a family of multimeric ATPases present in prokaryotes and eukaryotes (the Clp family), on the basis of its ability to remove the transposase from the DNA after recombination. Previously, ClpX has been shown to function with the ClpP peptidase in protein turnover. However, neither ClpP nor any other protease is required for disassembly of the transposase. The released MuA is not modified extensively, degraded, or irreversibly denatured, and is able to perform another round of recombination in vitro. We conclude that ClpX catalyzes the ATP-dependent release of MuA by promoting a transient conformational change in the protein and, therefore, can be considered a molecular chaperone. ClpX is important at the transition between the recombination and DNA replication steps of transposition in vitro; this function probably corresponds to the essential contribution of ClpX for Mu growth. Deletion analysis reveals that the sequence at the carboxyl terminus of MuA is important for disassembly by ClpX and can target MuA for degradation by ClpXP in vitro. These data contribute to the emerging picture that members of the Clp family are chaperones specifically suited for disaggregating proteins and are able to function with or without a collaborating protease.

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Disassembly of the Mu transposase tetramer by the ClpX chaperone.

Regulation of transposition in bacteria

Over the last ten years it has become increasingly apparent that turnover of particular proteins, under specific conditions, can play as central a role as the transcriptional and translational regulatory mechanisms. This review summarizes our current knowledge of this particular role of proteases responsible for the initial cleavages in most of these interesting degradative processes

Genetics of proteolysis in Escherichia coli

A genetic complementation system is described in which the complementing components are close together in a single linear DNA fragment; the complementation situation is temporary. This system is useful for providing transposition functions to transposition-defective transposons, since transposition functions act preferentially in cis. The basic procedure involves placing a transposition-defective transposon near the gene(s) for its transposition functions on a single DNA fragment. This fragment is introduced, here by general transduction, into a new host. The transposase acts in cis to permit the defective element to transpose from the introduced fragment into the recipient chromosome. The helper genes do not transpose and are lost by degradation and segregation. The method yields single insertion mutants that lack transposase and are not subject to further transposition or chromosome rearrangement. The general procedure is applicable to other sorts of transposable elements and could be modified for use in other genetic systems.

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Claudia Reich

Papers

Instability of transposase activity: Evidence from bacteriophage Mu DNA replication

Stoichiometric use of the transposase of bacteriophage Mu