Proteolysis in Escherichia coli serves to rid the cell of abnormal and misfolded proteins and to limit the time and amounts of availability of critical regulatory proteins. Most intracellular proteolysis is initiated by energy-dependent proteases, including Lon, ClpXP, and HflB; HflB is the only essential E. coli protease. The ATPase domains of these proteases mediate substrate recognition. Recognition elements in target are not well defined, but are probably not specific amino acid sequences. Naturally unstable protein substrates include the regulatory sigma factors for heat shock and stationary phase gene expression, sigma 32 and RpoS. Other cellular proteins serve as environmental sensors that modulate the availability of the unstable proteins to the proteases, resulting in rapid changes in sigma factor levels and therefore in gene transcription. Many of the specific proteases found in E. coli are well-conserved in both prokaryotes and eukaryotes, and serve critical functions in developmental systems.

Proteases and their targets in Escherichia coli.

https://www.cell.com/trends/biochemical-sciences/pdf/S0968-0004(96)10038-4.pdf

HSP100/Clp proteins: a common mechanism explains diverse functions

Proteases and chaperones together serve to maintain quahty control of cellular proteins. Both types of en­ zymes have as their substrates the variety of misfolded and partially folded proteins that arise from slow rates of folding or assembly, chemical or thermal stress, intrinsic structural instability, and biosynthetic errors. The pri­ mary function of classical chaperones, such as the Esch­ erichia coh DnaK/Hsp70 and its cochaperones, DnaJ and GrpE, and GroEL/Hsp60 and its cochaperone, GroES, is to modulate protein folding pathways, thereby prevent­ ing misfolding and aggregation, promoting refolding and proper assembly. Recent work has demonstrated that ATP-dependent proteases, as well as closely related pro­ teins, have intrinsic chaperone activity, suggesting that the initial steps in energy-dependent protein degradation may be similar to those of chaperone-dependent protein folding. The classical chaperones are also required for degradation of certain proteins in vivo, but we propose below that generally they affect proteolysis indirectly by maintaining proteins in soluble forms that would other­ wise aggregate and become inaccessible to proteases. In this review we present the evidence linking the activi­ ties of chaperones and proteases, and propose a general model for what can be thought of as a triage system for handling misfolded proteins in vivo, assuring swift re­ folding of proteins with functional potential and rapid degradation of irreversibly denatured or damaged pro­ teins.

/pdf/protein-quality-control-triage-by-chaperones-and-proteases-69nqdulikt.pdf

Protein quality control: triage by chaperones and proteases.

Escherichia coli K-12 F-prime factors, old and new.

The biology of restriction and anti-restriction.

Summary
We have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP-dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear-cut case of the versatile activity of polypeptides that form multi-component ATP-dependent proteases.

A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein

The timing of replication of an F'lac during the cell cycle of Escherichia coli B/r has been investigated at different growth rates to clarify the relationship of F factor replication to cell division and the replication of the bacterial chromosome. Cells of a lacZ — strain carrying an F'lac were separated according to their ages in an exponentially growing population after the culture was pulse labelled with a radioactive precursor of DNA and pulse induced for the synthesis of β-galactosidase. The amount of label incorporated at different cell ages reflects the state of replication of the bacteriial chromosome, while the amount of enzyme synthesized in response to a short period of induction is assumed to reflect the state of replication of the F'lac. The F'lac replicates at a time somewhat more than half way through the cell cycle at all growth rates investigated. This time is clearly distinguishable from the time of initiation of chromosomal replication at some of the growth rates studied, implying the existence of at least some different control elements in the replication of these two replicons. The regulation of F'lac replication has been further studied by following F'lac replication in temperature sensitive mutants, which are defective in the initiation of chromosomal replication at elevated temperatures.

Replication of the F'lac sex factor in the cell cycle of Escherichia coli.

Instability of transposase activity: Evidence from bacteriophage Mu DNA replication

A strong DNA gyrase-binding site (SGS) is located midway between the termini of the bacteriophage Mu genome and is required for efficient replicative transposition. We have proposed that the SGS promotes the efficient synapsis of the Mu prophage ends (an obligate early step in replicative transposition), and that it does so by helping to organize the prophage DNA into a supercoiled loop with the SGS at the apex of the loop and the prophage termini at the base. The positioning of the synapsing termini equidistant from the SGS is a key element in the proposed model. To test this proposal, we have constructed prophages with a second, internal right end and asked whether the natural, external right end or the internal right end is used for synapsis with the left end in the presence and absence of the SGS. In the presence of the central SGS, the natural, or outside, right end was used exclusively and very efficiently. In the absence of the central SGS, the internal right end was used preferentially and inefficiently: the efficiency of transposition decreased with increasing distance between the internal right end and the left end. Repositioning the SGS midway between the left end and an internal right end allowed highly efficient use of the internal right end. These results support a model in which gyrase can influence long-range DNA interactions to promote efficient synapsis of Mu prophage ends.

Martin L. Pato

Papers

A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein

Replication of the F'lac sex factor in the cell cycle of Escherichia coli.

Instability of transposase activity: Evidence from bacteriophage Mu DNA replication

The Mu strong gyrase-binding site promotes efficient synapsis of the prophage termini.

Replication of mini-Mu prophage DNA.