Agnieszka Szalewska-Pałasz

The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase.

Some promoters, including the DmpR-controlled ςN-dependent Po promoter, are effectively rendered silent in cells lacking the nutritional alarmone (p)ppGpp. Here we demonstrate that four mutations within the housekeeping ςD-factor can restore ςN-dependent Po transcription in the absence of (p)ppGpp. Using both in vitro and in vivotranscription competition assays, we show that all the four ςD mutant proteins are defective in their ability to compete with ςN for available core RNA polymerase and that the magnitude of the defect reflects the hierarchy of restoration of transcription from Po in (p)ppGpp-deficient cells. Consistently, underproduction of ςD or overproduction of the anti-ςD protein Rsd were also found to allow (p)ppGpp-independent transcription from the ςN-Po promoter. Together with data from the direct effects of (p)ppGpp on ςN-dependent Po transcription and ς-factor competition, the results support a model in which (p)ppGpp serves as a master global regulator of transcription by differentially modulating alternative ς-factor competition to adapt to changing cellular nutritional demands.

Antagonistic regulation of Escherichia coli ribosomal RNA rrnB P1 promoter activity by GreA and DksA.

The Escherichia coli proteins DksA, GreA, and GreB are all structural homologs that bind the secondary channel of RNA polymerase (RNAP) but are thought to act at different levels of transcription. DksA, with its co-factor ppGpp, inhibits rrnB P1 transcription initiation, whereas GreA and GreB activate RNAP to cleave back-tracked RNA during elongational pausing. Here, in vivo and in vitro evidence reveals antagonistic regulation of rrnB P1 transcription initiation by Gre factors (particularly GreA) and DksA; GreA activates and DksA inhibits. DksA inhibition is epistatic to GreA activation. Both modes of regulation are ppGpp-independent in vivo but DksA inhibition requires ppGpp in vitro. Kinetic experiments and studies of rrnB P1-RNA polymerase complexes suggest that GreA mediates conformational changes at an initiation step in the absence of NTP substrates, even before DksA acts. GreA effects on rrnB P1 open complex conformation reveal a new feature of GreA distinct from its general function in elongation. Our findings support the idea that a balance of the interactions between the three secondary channel-binding proteins and RNAP can provide a new mode for regulating transcription.

ppGpp inhibits the activity of Escherichia coli DnaG primase

DNA primase is an enzyme required for replication of both chromosomes and vast majority of plasmids. Guanosine tetra- and penta-phosphate (ppGpp and pppGpp, respectively) are alarmones of the bacterial stringent response to starvation and stress conditions, and act by modulation of the RNA polymerase activity. Recent studies indicated that the primase-catalyzed reaction is also inhibited by (p)ppGpp in Bacillus subtilis, where a specific regulation of DNA replication elongation, the replication fork arrest, was discovered. Although in Escherichia coli such a replication regulation was not reported to date, here we show that E. coli DnaG primase is directly inhibited by ppGpp and pppGpp. However, contrary to the B. subtilis primase response to the stringent control alarmones, the E, coli DnaG was inhibited more efficiently by ppGpp than by pppGpp.

Properties of RNA Polymerase Bypass Mutants IMPLICATIONS FOR THE ROLE OF ppGpp AND ITS CO-FACTOR DksA IN CONTROLLING TRANSCRIPTION DEPENDENT ON σ54

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain σ70-dependent promoters. A number of promoters that are dependent on alternative σ-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived σ54-dependent Po promoter and several other σ54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro σ54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the β- and β′-subunits of core RNA polymerase that allow high levels of transcription from the σ54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with σ-factors to favor σ54-holoenzyme formation over that with σ70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of σ54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent σ70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative σ-factors is controlled to meet cellular requirements.

The mechanism of the stringent control of lambda plasmid DNA replication.

Lambda plasmid DNA replication is inhibited in amino acid‐starved wild type Escherichia coli strains (stringent response) but not in amino acid‐starved relA mutants (relaxed response). This replication is perpetuated by the replication complex containing the lambda O protein (which is protected from proteases by other elements of the complex) and inherited by one of two daughter copies after a replication round. Since a fraction of stable lambda O protein was observed in relA‐ and relA+ strains, and negative regulation by the lambda Cro repressor does not seem to be important in the stringent or relaxed response of lambda plasmid replication to amino acid starvation, the inhibition of lambda plasmid replication in amino acid‐starved wild type strains was investigated. lambda plasmids were unable to replicate in amino acid‐starved relA‐ bacteria treated with rifampicin. Moreover, transcription from pR, which produces mRNA for replication protein synthesis and serves as transcriptional activation of ori lambda, was significantly decreased during the stringent response as well as in non‐starved cells containing increased levels of ppGpp. However, it was little or totally not affected by the relaxed response. The replacement of pR with plac (which is known to be uninhibited by ppGpp) in a lambda plasmid resulted in its DNA replication during relaxed and stringent responses as well as during overproduction of ppGpp in unstarved bacteria. We conclude that ppGpp‐mediated inhibition of transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid DNA replication in amino acid‐starved wild type strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential inhibition of transcription from σ70- and σ32-dependent promoters by rifampicin

Rifampicin is an antibiotic which binds to the β subunit of prokaryotic RNA polymerases and prevents initiation of transcription. It was found previously that production of heat shock proteins in Escherichia coli cells after a shift from 30°C to 43°C is not completely inhibited by this antibiotic. Here we demonstrate that while activity of a p L‐lacZ fusion (p L is a σ70‐dependent promoter) in E. coli cells is strongly inhibited by rifampicin, a p groE‐lacZ fusion, whose activity is dependent on the σ32 factor, retains significant residual activity even at relatively high rifampicin concentrations. Differential sensitivity to this antibiotic of RNA polymerase holoenzymes containing either the σ70 or the σ32 subunit was confirmed in vitro. Since the effects of an antibiotic that binds to the β subunit can be modulated by the presence of either the σ70 or the σ32 subunit in the holoenzyme, it is tempting to speculate that binding of various σ factors to the core of RNA polymerase results in different conformations of particular holoenzymes, including changes in the core enzyme.

Genetic response to metabolic fluctuations: correlation between central carbon metabolism and DNA replication in Escherichia coli

BackgroundUntil now, the direct link between central carbon metabolism and DNA replication has been demonstrated only in Bacillus. subtilis. Therefore, we asked if this is a specific phenomenon, characteristic for this bacterium and perhaps for its close relatives, or a more general biological rule.ResultsWe found that temperature-sensitivity of mutants in particular genes coding for replication proteins could be suppressed by deletions of certain genes coding for enzymes of the central carbon metabolism. Namely, the effects of dnaA46(ts) mutation could be suppressed by dysfunction of pta or ackA, effects of dnaB(ts) by dysfunction of pgi or pta, effects of dnaE486(ts) by dysfunction of tktB, effects of dnaG(ts) by dysfunction of gpmA, pta or ackA, and effects of dnaN159(ts) by dysfunction of pta or ackA. The observed suppression effects were not caused by a decrease in bacterial growth rate.ConclusionsThe genetic correlation exists between central carbon metabolism and DNA replication in the model Gram-negative bacterium, E. coli. This link exists at the steps of initiation and elongation of DNA replication, indicating the important global correlation between metabolic status of the cell and the events leading to cell reproduction.

Transcriptional activation of the origin of coliphage lambda DNA replication is regulated by the host DnaA initiator function.

The initiator of phage lambda DNA replication, the lambda O protein, is considered to be an analogue of the initiator of DNA replication (DnaA) of its host, Escherichia coli. Both specifically recognize their origins of replication, ori lambda and oriC, respectively, and organize the assembly of specific replication complexes. However, DnaA has an additional activation function, acting on oriC-proximal DnaA-boxes, and regulating transcription initiated at promoters in and around oriC. Here, we demonstrate that lambda plasmid replication can be synchronized by a temperature shift-down that caused renaturation of the previously denatured DnaAts protein. Moreover, we show that elimination of the activating DnaA function affects transcriptional activation at ori lambda. DnaA may act by binding to DnaA-boxes, situated around the lambda pR promoter; there are no such sequences in ori lambda. Our results being to explain in molecular terms why lambda plasmid replication is DnaA-dependent [Kur et al., J. Mol. Biol. 198 (1987) 203-210] and why the initiation of phage lambda DNA replication is blocked (in E. coli devoid of prophage Rac) after inactivation of DnaA [Wegrzyn et al., Genetics (1995) in press].

Transcription from bacteriophage λ pR promoter is regulated independently and antagonistically by DksA and ppGpp

The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage λ major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksA-mediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in λ phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation.

INTERACTION OF THE ESCHERICHIA COLI DNAA PROTEIN WITH BACTERIOPHAGE LAMBDA DNA

Abstract Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage λ DNA demonstrated differential binding of DnaA along the whole λ DNA. Interaction of DnaA with the λ replication region (from the promoter pR to the origin of replication, oriλ) demonstrated a strong binding of DnaA to the region around the po promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the λ DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.