The heat-shock response (HSR), a universal cellular response to heat, is crucial for cellular adaptation. In Escherichia coli, the HSR is mediated by the alternative factor, 32 . To determine its role, we used genome-wide expression analysis and promoter validation to identify genes directly regulated by 32 and screened ORF overexpression libraries to identify 32 inducers. We triple the number of genes validated to be transcribed by 32 and provide new insights into the cellular role of this response. Our work indicates that the response is propagated as the regulon encodes numerous global transcriptional regulators, reveals that 70 holoenzyme initiates from 12% of 32 promoters, which has important implications for global transcriptional wiring, and identifies a new role for the response in protein homeostasis, that of protecting complex proteins. Finally, this study suggests that the response protects the cell membrane and responds to its status: Fully 25% of 32 regulon members reside in the membrane and alter its functionality; moreover, a disproportionate fraction of overexpressed proteins that induce the response are membrane localized. The intimate connection of the response to the membrane rationalizes why a major regulator of the response resides in that cellular compartment.[Keywords: Heat-shock response; 32; transcription; microarray] Supplemental material is available at http://www.genesdev.org. Received March 13, 2006; revised version accepted April 25, 2006. When cells are shifted from low to high temperature, synthesis of the heat-shock proteins (hsps) is rapidly and selectively induced. The heat-shock response (HSR), was first identified by Ritossa (1963), who showed that exposure to heat lead to transient changes in the puffing pattern of salivary chromosomes in Drosophila; Tissieres et al. (1974) demonstrated that these changes reflected the transient induction of several proteins. Initially, hsp function was unclear; however, experiments in several organisms revealed that many hsps were chaperones that promote protein folding (Pelham 1986;Beckmann et al. 1990;Gaitanaris et al. 1990;Skowyra et al. 1990). These studies not only suggested that a major function of the HSR is to maintain the protein folding state of the cell, but also indicated that some of these chaperones, such as Hsp70 and Hsp90, are present in all organisms Craig 1984, 1987). Thus, both the HSR and some hsps are universally conserved among organisms.In Escherichia coli, 32 , an alternative factor, controls the HSR by directing RNA polymerase to transcribe hsps (Yamamori and Yura 1980;Grossman et al. 1984;Taylor et al. 1984;Cowing et al. 1985). Synthesis of hsps is induced upon temperature upshift and repressed upon temperature downshift (Lemaux et al. 1978;Yamamori et al. 1978;Straus et al. 1987 Straus et al. , 1989Taura et al. 1989), thereby allowing a rapid cellular response to changes in temperature.32 is controlled by negative feedback loops controlling its activity (Straus et al. 1989;Blaszczak et al. 1999) and stabili...

Summary Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli by removing transcription roadblocks. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This arrest requires active transcription, and is alleviated by RNA polymerase mutants that compensate for DksA activity. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA damage response and recruits the main recombination protein RecA. This novel function of DksA is independent of its transcription initiation activity, but requires its less studied transcription elongation activity. Finally, GreA/B elongation factors also prevent replication arrest during nutrient stress. We conclude that transcription elongation factors alleviate fundamental conflicts between replication and transcription, thereby protecting replication fork progression and DNA integrity.

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

No abstract

While we have recently constructed a functional reversal of the β-oxidation cycle as a platform for the production of fuels and chemicals by engineering global regulators and eliminating native fermentative pathways, the system-level approach used makes it difficult to determine which of the many deregulated enzymes are responsible for product synthesis. This, in turn, limits efforts to fine-tune the synthesis of specific products and prevents the transfer of the engineered pathway to other organisms. In the work reported here, we overcome the aforementioned limitations by using a synthetic biology approach to construct and functionally characterize a reversal of the β-oxidation cycle. This was achieved through the in vitro kinetic characterization of each functional unit of the core and termination pathways, followed by their in vivo assembly and functional characterization. With this approach, the four functional units of the core pathway, thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase, were purified and kinetically characterized in vitro. When these four functional units were assembled in vivo in combination with thioesterases as the termination pathway, the synthesis of a variety of 4-C carboxylic acids from a one-turn functional reversal of the β-oxidation cycle was realized. The individual expression and modular construction of these well-defined core components exerted the majority of control over product formation, with only highly selective termination pathways resulting in shifts in product formation. Further control over product synthesis was demonstrated by overexpressing a long-chain thiolase that enables the operation of multiple turns of the reversal of the β-oxidation cycle and hence the synthesis of longer-chain carboxylic acids. The well-defined and self-contained nature of each functional unit makes the engineered reversal of the β-oxidation cycle "chassis neutral" and hence transferrable to the host of choice for efficient fuel or chemical production.

Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.

The RecQ-helicase family is widespread, is highly conserved, and includes human orthologs that suppress genomic instability and cancer. In vivo, some RecQ homologs promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that, in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promotes this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologs.

No abstract