DnaA-ATP acts as a molecular switch to control levels of ribonucleotide reductase expression in Escherichia coli: DnaA and ribonucleotide reductase expression (original) (raw)
Related papers
Molecular Microbiology, 2010
Ribonucleotide reductase (RNR) is the bottleneck enzyme in the synthesis of dNTPs 2 required for DNA replication. In order to avoid the mutagenic effects of imbalances in dNTPs 3 the amount and activity of RNR enzyme in the cell is tightly regulated. RNR expression from 4 the nrdAB operon is thus coupled to coincide with the initiation of DNA replication. However 5 the mechanism for the coordination of gene transcription and DNA replication remains to be 6 elucidated. The timing and synchrony of DNA replication initiation in Escherichia coli is 7 controlled in part by the binding of the DnaA protein to the origin of replication. DnaA is also 8 a transcription factor of the nrdAB operon and could thus be the link between these two 9 processes. Here we show that RNA polymerase can form a stable transcription initiation 10 complex at the nrdAB promoter by direct interaction with the far upstream sites required for 11 the timing of expression as a function of DNA replication. In addition, we show that the 12 binding of DnaA on the promoter can either activate or repress transcription as a function of 13 its concentration and its nucleotide-bound state. However, transcription regulation by DnaA 14 does not significantly affect the timing of expression of RNR from the nrdAB operon.
The EMBO Journal, 2006
We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the b-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda. The b-subunit acts as a matchmaker between Hda and DnaA. Chain elongation of DNA requires a sufficient supply of deoxyribonucleotides (dNTPs), which are produced by ribonucleotide reductase (RNR). We present evidence suggesting that the molecular switch from ATP-DnaA to ADP-DnaA is a critical step coordinating DNA replication with increased deoxyribonucleotide synthesis. Characterization of dnaA and dnaN mutations that result in a constitutively high expression of RNR reveal this mechanism. We propose that the nucleotide bound state of DnaA regulates the transcription of the genes encoding ribonucleotide reductase (nrdAB). Accordingly, the conversion of ATP-DnaA to ADP-DnaA after initiation and loading of the b-subunit DnaN would allow increased nrdAB expression, and consequently, coordinated RNR synthesis and DNA replication during the cell cycle.
A Nucleotide Switch in the Escherichia coli DnaA Protein Initiates Chromosomal Replication
Journal of Biological Chemistry, 2002
The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp-and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.
Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage
Molecular Microbiology, 2007
All organisms that synthesize their own DNA have evolved mechanisms for maintaining a constant DNA/cell mass ratio independent of growth rate. The DNA/cell mass ratio is a central parameter in the processes controlling the cell cycle. The co-ordination of DNA replication with cell growth involves multiple levels of regulation. DNA synthesis is initiated at specific sites on the chromosome termed origins of replication, and proceeds bidirectionally to elongate and duplicate the chromosome. These two processes, initiation and elongation, therefore determine the total rate of DNA synthesis in the cell. In Escherichia coli, initiation depends on the DnaA protein while elongation depends on a multiprotein replication factory that incorporates deoxyribonucleotides (dNTPs) into the growing DNA chain. The enzyme ribonucleotide reductase (RNR) is universally responsible for synthesizing the necessary dNTPs. In this review we examine the role RNR plays in regulating the total rate of DNA synthesis in E. coli and, hence, in maintaining constant DNA/cell mass ratios during normal growth and under conditions of DNA stress.
The EMBO Journal, 1999
The ATP-bound but not the ADP-bound form of DnaA protein is active for replication initiation at the Escherichia coli chromosomal origin. The hydrolysis of ATP bound to DnaA is accelerated by the sliding clamp of DNA polymerase III loaded on DNA. Using a culture of randomly dividing cells, we now have evidence that the cellular level of ATP-DnaA is repressed to only~20% of the total DnaA molecules, in a manner depending on DNA replication. In a synchronized culture, the ATP-DnaA level showed oscillation that has a temporal increase around the time of initiation, and decreases rapidly after initiation. Production of ATP-DnaA depended on concomitant protein synthesis, but not on SOS response, Dam or SeqA. Regeneration of ATP-DnaA from ADP-DnaA was also observed. These results indicate that the nucleotide form shifts of DnaA are tightly linked with an epistatic cell cycle event and with the chromosomal replication system.
Molecular Microbiology, 2002
Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli of 70 bp s-1. In contrast to this difference in polymerization, the dNTP pool is about 10 times smaller than the NTP pool (Pato, 1979). This discrepancy was observed very early on by Werner (1971), who asked how the intracellular concentration of dNTP could be sufficient to support the observed rate of DNA replication. Besides this difference in pools, dNTPs are highly specialized molecules, as they have few roles outside DNA replication, and this functionality is highly localized at only a few intracellular sites. In a work on the isolation of a DNA replication system bound to membrane in rat liver and hepatomes, Baril et al. (1974) demonstrated the incorporation of thymidine in their in vitro system and were the first to propose a multienzyme replication complex in which DNA polymerase II and at least three enzymes involved in the dNTP biosynthesis take part. Since then, many experiments have demonstrated the presence of some of the enzymes involved in dNTP synthesis in a multienzyme complex in both prokaryotic and eukaryotic cells (reviewed by Mathews, 1993). Three observations suggest a multienzyme complex for dNTP biosynthesis associated with the DNA replication apparatus: (i) the incorporation of radiolabelled thymidine into DNA reaches its maximal rate before the pool of dTTP is fully labelled (Werner, 1971; Pato, 1979); (ii) permeabilized bacterial cells incorporate deoxyribonucleoside diphosphates into DNA more efficiently than the corresponding triphosphates; and (iii) inhibition of nucleoside diphosphate kinase inhibits direct incorporation of dNTP into DNA in permeabilized cells (Reddy and Mathews, 1978). This model of a multienzyme complex also suggests that the transfer of dNTP to DNA polymerase is facilitated by channelling and com
The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein
Frontiers in Microbiology
This review summarizes the mechanisms of the initiator protein DnaA in replication initiation and its regulation in Escherichia coli. The chromosomal origin (oriC) DNA is unwound by the replication initiation complex to allow loading of DnaB helicases and replisome formation. The initiation complex consists of the DnaA protein, DnaAinitiator-associating protein DiaA, integration host factor (IHF), and oriC, which contains a duplex-unwinding element (DUE) and a DnaA-oligomerization region (DOR) containing DnaA-binding sites (DnaA boxes) and a single IHF-binding site that induces sharp DNA bending. DiaA binds to DnaA and stimulates DnaA assembly at the DOR. DnaA binds tightly to ATP and ADP. ATP-DnaA constructs functionally different sub-complexes at DOR, and the DUE-proximal DnaA sub-complex contains IHF and promotes DUE unwinding. The first part of this review presents the structures and mechanisms of oriC-DnaA complexes involved in the regulation of replication initiation. During the cell cycle, the level of ATP-DnaA level, the active form for initiation, is strictly regulated by multiple systems, resulting in timely replication initiation. After initiation, regulatory inactivation of DnaA (RIDA) intervenes to reduce ATP-DnaA level by hydrolyzing the DnaA-bound ATP to ADP to yield ADP-DnaA, the inactive form. RIDA involves the binding of the DNA polymerase clamp on newly synthesized DNA to the DnaA-inactivator Hda protein. In datA-dependent DnaA-ATP hydrolysis (DDAH), binding of IHF at the chromosomal locus datA, which contains a cluster of DnaA boxes, results in further hydrolysis of DnaA-bound ATP. SeqA protein inhibits untimely initiation at oriC by binding to newly synthesized oriC DNA and represses dnaA transcription in a cell cycle dependent manner. To reinitiate DNA replication, ADP-DnaA forms oligomers at DnaA-reactivating sequences (DARS1 and DARS2), resulting in the dissociation of ADP and the release of nucleotide-free apo-DnaA, which then binds ATP to regenerate ATP-DnaA. In vivo, DARS2 plays an important role in this process and its activation is regulated by timely binding of IHF to DARS2 in the cell cycle. Chromosomal locations of DARS sites are optimized for the strict regulation for timely replication initiation. The last part of this review describes how DDAH and DARS regulate DnaA activity.
Journal of Biological Chemistry
The rrnB P1 promoter of Escherichia coli (starting sequence C"4-A"S-C"2-C"1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Rea. 16,9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3+ " mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1+" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-ternary complexes carry u factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3+ ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
Proceedings of the National Academy of Sciences, 2004
A strain of Escherichia coli missing three members of the thioredoxin superfamily, thioredoxins 1 and 2 and glutaredoxin 1, is unable to grow, a phenotype presumed to be due to the inability of cells to reduce the essential enzyme ribonucleotide reductase. Two classes of mutations can restore growth to such a strain. First, we have isolated a collection of mutations in the gene for the protein glutaredoxin 3 that suppress the growth defect. Remarkably, all eight independent mutations alter the same amino acid, methionine-43, changing it to valine, isoleucine, or leucine. From the position of the amino acid changes and their effects, we propose that these alterations change the protein so that its properties are closer to those of glutaredoxin 1. The second means of suppressing the growth defects of the multiply mutant strain was by mutations in the DNA replication genes, dnaA and dnaN. These mutations substantially increase the expression of ribonucleotide reductase, most likely by altering the interaction of the regulatory protein DnaA with the ribonucleotide reductase promoter. Our results suggest that this increase in the concentration of ribonucleotide reductase in the cell allows more effective interaction with glutaredoxin 3, thus restoring an effective pool of deoxyribonucleotides. Our studies present direct evidence that ribonucleotide reductase is the only essential enzyme that requires the three reductive proteins missing in our strains. Our results also suggest an unexpected regulatory interaction between the DnaA and DnaN proteins.
Nucleic acids research, 2017
In Escherichia coli, the level of the ATP-DnaA initiator is increased temporarily at the time of replication initiation. The replication origin, oriC, contains a duplex-unwinding element (DUE) flanking a DnaA-oligomerization region (DOR), which includes twelve DnaA-binding sites (DnaA boxes) and the DNA-bending protein IHF-binding site (IBS). Although complexes of IHF and ATP-DnaA assembly on the DOR unwind the DUE, the configuration of the crucial nucleoprotein complexes remains elusive. To resolve this, we analyzed individual DnaA protomers in the complex and here demonstrate that the DUE-DnaA-box-R1-IBS-DnaA-box-R5M region is essential for DUE unwinding. R5M-bound ATP-DnaA predominantly promotes ATP-DnaA assembly on the DUE-proximal DOR, and R1-bound DnaA has a supporting role. This mechanism might support timely assembly of ATP-DnaA on oriC. DnaA protomers bound to R1 and R5M directly bind to the unwound DUE strand, which is crucial in replication initiation. Data from in vivo e...