Chi Zen Lu

rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli.

In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E. The sigma 32‐regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins. To further understand this response, we located the rpoE gene at 55.5′ and analyzed the role of sigma E. sigma E is required at high temperature, and controls the transcription of at least 10 genes. Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet. sigma E controls its own transcription from a sigma E‐dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity. Indeed, under steady‐state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell. However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE. Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal.

Lack of a Robust Unfoldase Activity Confers a Unique Level of Substrate Specificity to the Universal AAA Protease FtsH

FtsH, a member of the AAA family of proteins, is the only membrane ATP-dependent protease universally conserved in prokaryotes, and the only essential ATP-dependent protease in Escherichia coli. We investigated the mechanism of degradation by FtsH. Other well-studied ATP-dependent proteases use ATP to unfold their substrates. In contrast, both in vitro and in vivo studies indicate that degradation by FtsH occurs efficiently only when the substrate is a protein of low intrinsic thermodynamic stability. Because FtsH lacks robust unfoldase activity, it is able to use the protein folding state of substrates as a criterion for degradation. This feature may be key to its role in the cell and account for its ubiquitous distribution among prokaryotic organisms.

Structural Features Required for the Interaction of the Hsp70 Molecular Chaperone DnaK with Its Cochaperone DnaJ

Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.

A Coiled-Coil from the RNA Polymerase β′ Subunit Allosterically Induces Selective Nontemplate Strand Binding by σ70

Abstract For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the −10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the β′ subunit (aa 262–309) induces σ 70 to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the β′ coiled-coil and region 2.2 of σ 70 promotes an allosteric transition that allows σ 70 to selectively recognize the nontemplate strand. As the β′ 262–309 peptide can function with the previously crystallized portion of σ 70 , nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

Binding of the Initiation Factor σ70 to Core RNA Polymerase Is a Multistep Process

Abstract The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation. To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and σ 70 , the Escherichia coli initiation factor. We identified nine mutationally verified interaction sites between σ 70 and specific domains of RNA polymerase and provide evidence that σ 70 and RNA polymerase interact in at least a two-step process. We propose that a cycle of changes in the interface of σ 70 with core RNA polymerase is associated with progression through the process of transcription initiation.

Analysis of σ32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response

Protein quality control is accomplished by inducing chaperones and proteases in response to an altered cellular folding state. In Escherichia coli, expression of chaperones and proteases is positively regulated by σ32. Chaperone-mediated negative feedback control of σ32 activity allows this transcription factor to sense the cellular folding state. We identified point mutations in σ32 altered in feedback control. Surprisingly, such mutants are resistant to inhibition by both the DnaK/J and GroEL/S chaperones in vivo and also show dramatically increased stability. Further characterization of the most defective mutant revealed that it has almost normal binding to chaperones and RNA polymerase and is competent for chaperone-mediated inactivation in vitro. We suggest that the mutants identify a regulatory step downstream of chaperone binding that is required for both inactivation and degradation of σ32.