Growth Rate Regulation and Control of Initiation of DNA Replication in Escherichia Coli PDF Download
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Author: Anne Eliane Chiaramello
Publisher:
ISBN:
Category : DNA
Languages : en
Pages : 300
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Book Description
The initiation of DNA synthesis at the chromosomal replication origin, oriC, in Escherichia coli involves an RNA polymerase-mediated step. The level of synthesis of transcripts moving counterclockwise toward oriC is controlled at two promoters, P1 (asnC) and P2 (mioC), and at two transcription terminator regions, T1 and T2. As shown by S1 mapping, termination at the T2 region occurs to the right of oriC at nucleotides 297-299 and 306-310, while major termination events in the T1 region occur in and near the mioC promoter. The majority of transcripts entering oriC originates from the mioC promoter. Transcription from the mioC promoter has been shown to enhance the frequency of initiation of DNA replication of oriC containing plasmids, and to stabilize these plasmids in the host cells. The mioC promoter, which is stringently controlled, is also growth rate regulated. The amount of mioC transcripts relative to the amount of total RNA was inversely correlated with growth rate. This transcript is characterized by a short half-life (1.5 min). The mioC promoter, which contains a DnaA protein binding site, was much less susceptible to repression by DnaA protein when located in the chromosome, than when located in a plasmid. Only a very high concentration of DnaA protein repressed the mioC promoter. The DnaA protein, which is required for initiation of DNA replication from oriC, is growth rate regulated. As shown by RNase protection, this regulation is exerted at the transcriptional level, affecting both promoters, dnaAp1 and dnaAp2. Transcription from these two promoters is also stringently controlled. The amount of DnaA protein in spoT mutants, which are deficient in ppGpp pyrophosphorylase activity, decreases as the severity in the mutation increases. Thus, the intracellular concentration of ppGpp influences the expression of the dnaA gene. In conclusion, the growth rate regulation and stringent control of the dnaA gene suggest that one way in which DNA replication is coordinated with the growth rate is via ppGpp synthesis at the ribosome.
Author: Anne Eliane Chiaramello
Publisher:
ISBN:
Category : DNA
Languages : en
Pages : 300
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Book Description
The initiation of DNA synthesis at the chromosomal replication origin, oriC, in Escherichia coli involves an RNA polymerase-mediated step. The level of synthesis of transcripts moving counterclockwise toward oriC is controlled at two promoters, P1 (asnC) and P2 (mioC), and at two transcription terminator regions, T1 and T2. As shown by S1 mapping, termination at the T2 region occurs to the right of oriC at nucleotides 297-299 and 306-310, while major termination events in the T1 region occur in and near the mioC promoter. The majority of transcripts entering oriC originates from the mioC promoter. Transcription from the mioC promoter has been shown to enhance the frequency of initiation of DNA replication of oriC containing plasmids, and to stabilize these plasmids in the host cells. The mioC promoter, which is stringently controlled, is also growth rate regulated. The amount of mioC transcripts relative to the amount of total RNA was inversely correlated with growth rate. This transcript is characterized by a short half-life (1.5 min). The mioC promoter, which contains a DnaA protein binding site, was much less susceptible to repression by DnaA protein when located in the chromosome, than when located in a plasmid. Only a very high concentration of DnaA protein repressed the mioC promoter. The DnaA protein, which is required for initiation of DNA replication from oriC, is growth rate regulated. As shown by RNase protection, this regulation is exerted at the transcriptional level, affecting both promoters, dnaAp1 and dnaAp2. Transcription from these two promoters is also stringently controlled. The amount of DnaA protein in spoT mutants, which are deficient in ppGpp pyrophosphorylase activity, decreases as the severity in the mutation increases. Thus, the intracellular concentration of ppGpp influences the expression of the dnaA gene. In conclusion, the growth rate regulation and stringent control of the dnaA gene suggest that one way in which DNA replication is coordinated with the growth rate is via ppGpp synthesis at the ribosome.
Author:
Publisher:
ISBN:
Category : DNA
Languages : en
Pages : 260
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Book Description
The initiation of DNA synthesis at the chromosomal replication origin, oriC, in Escherichia coli involves an RNA polymerase-mediated step. The level of synthesis of transcripts moving counterclockwise toward oriC is controlled at two promoters, P1 (asnC) and P2 (mioC), and at two transcription terminator regions, T1 and T2. As shown by S1 mapping, termination at the T2 region occurs to the right of oriC at nucleotides 297-299 and 306-310, while major termination events in the T1 region occur in and near the mioC promoter. The majority of transcripts entering oriC originates from the mioC promoter. Transcription from the mioC promoter has been shown to enhance the frequency of initiation of DNA replication of oriC containing plasmids, and to stabilize these plasmids in the host cells. The mioC promoter, which is stringently controlled, is also growth rate regulated. The amount of mioC transcripts relative to the amount of total RNA was inversely correlated with growth rate. This transcript is characterized by a short half-life (1.5 min). The mioC promoter, which contains a DnaA protein binding site, was much less susceptible to repression by DnaA protein when located in the chromosome, than when located in a plasmid. Only a very high concentration of DnaA protein repressed the mioC promoter. The DnaA protein, which is required for initiation of DNA replication from oriC, is growth rate regulated. As shown by RNase protection, this regulation is exerted at the transcriptional level, affecting both promoters, dnaAp1 and dnaAp2. Transcription from these two promoters is also stringently controlled. The amount of DnaA protein in spoT mutants, which are deficient in ppGpp pyrophosphorylase activity, decreases as the severity in the mutation increases. Thus, the intracellular concentration of ppGpp influences the expression of the dnaA gene. In conclusion, the growth rate regulation and stringent control of the dnaA gene suggest that one way in which DNA replication is coordinated with the growth rate is via ppGpp synthesis at the ribosome.
Author: Monika Glinkowska
Publisher: Springer
ISBN: 3319105337
Category : Science
Languages : en
Pages : 56
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Book Description
This work describes the current knowledge of biochemical mechanisms regulating initiation of DNA replication in Escherichia coli, which focuses on the control of activity of the DnaA protein. Examples of direct linkages between DNA replication and other cellular processes are provided. In addition, similarities of the mechanisms of regulation of DNA replication operating in prokaryotic and eukaryotic cells are identified, and implications for understanding more complex processes, like carcinogenesis are suggested. Studies of recent years provided evidence that regulation of DNA replication in bacteria is more complex than previously anticipated. Multiple layers of control seem to ensure coordination of this process with the increase of cellular mass and the division cycle. Metabolic processes and membrane composition may serve as points where integration of genome replication with growth conditions occurs. It is also likely that coupling of DNA synthesis with cellular metabolism may involve interactions of replication proteins with other macromolecular complexes, responsible for various cellular processes. Thus, the exact set of factors participating in triggering the replication initiation may differ depending on growth conditions. Therefore, understanding the regulation of DNA duplication requires placing this process in the context of the current knowledge on bacterial metabolism, as well as cellular and chromosomal structure. Moreover, in both Escherichia coli and eukaryotic cells, replication initiator proteins were shown to play other roles in addition to driving the assembly of replication complexes, which constitutes another, yet not sufficiently understood, layer of coordinating DNA replication with the cell cycle.
Author: Michael Dalbey
Publisher:
ISBN:
Category : DNA.
Languages : en
Pages : 378
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Author: Dongyang Li
Publisher:
ISBN:
Category :
Languages : en
Pages : 178
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Book Description
The defining feature of living organisms is their capacity to reproduce and pass on the genetic information so that their progeny can flourish. For bacteria, reproduction is a feat by itself--Escherichia coli cells cultured in optimal conditions grow rapidly and divide about every 20 minutes. In other words, the cell has to replicate all cellular contents, and be ready to divide evenly into two daughter cells within this 20 minutes. Biosynthesis of new cellular materials, e.g. proteins, nucleic acis, lipids and other metabolites accumulate and roughly doubles after every generation. Notably, the deoxyribonucleic acid (DNA) encodes genetic informationand needs to be duplicated in order to faithfully pass on this information to the progeny. This process of DNA replication in the cell needs to dynamically adapt to fluctuation in growth condition and cellular physiology. Such coordination is controlled at the first step of replcation--the initiation of replication. In this thesis, I presented the development of methods for measuring DNA replication duration (replication period), the quantitative relationship between DNA replication and cell size as well as the mechanism of replication initiation. DNA replication measurement laid the foundation of studying the quantitative relationship between cell size and DNA replication. A general growth law was proposed to describe cell size regulation in light of three physiological variables including biosynthesis rate, cell cycle progression and replicaiton initiation. Of the three variables, the mass when cell initiates replication (initiation mass) remains invariant despite a wide spectrum of antibiotics or growth limitation challenge. This invariant initiation mass called into question about the mechanism of initiation to achieve such constancy. We proposed a simple threshold model to explain how cells can maintain a invariant initiation mass by regulating the expression of initiation regulators (initiators). The initiation mass is inversely proportional to the initiator levels, which is held constant. Experimental evidence was provided to test our model prediction.
Author: Ole Maaløe
Publisher:
ISBN:
Category : Bacteria
Languages : en
Pages : 306
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Author:
Publisher:
ISBN: 9780815332183
Category : Cells
Languages : en
Pages : 0
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Author: C. W. Lycett
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Thomas David Daws
Publisher:
ISBN:
Category :
Languages : en
Pages : 272
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Book Description
Author: Victoria Goehner Newman
Publisher:
ISBN:
Category : DNA replication
Languages : en
Pages : 492
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Book Description
The timing of initiation of DNA replication from oriC in E. coli is regulated by the concentration of the DnaA protein. Increases in the intracellular DnaA protein concentration over 1.5 fold normal levels causes asynchronous timing of initiation of DNA replication. The regulators of the dnaA operon must keep the DnaA concentration within tight limits yet be able to respond to rapidly changing environmental conditions. Here it is shown that Fis and DnaA proteins interact, in conjunction with other factors, to regulate the expression of dnaA. Binding of DnaA to the DnaA box in the dnaA promoter region is destabilized by the binding of Fis to its binding site in dnaAp2. However, DnaA94, containing only the C-terminal 94 amino acids of the DnaA protein and retaining specific binding activity to the DnaA box of the dnaA promoter region, is not destabilized by Fis binding. DNase I and copper-phenanthroline (Cu-op) footprinting studies of DnaA and DnaA94 support the existence of a second weak DnaA box which abuts the DNase I and Cu-op footprints of Fis in the dnaAp2 promoter. We determined that the GC-rich region of the dnaAp2 promoter is analogous to the discriminator sequence (DS) of rRNA promoters, in that the GC-rich sequence mediates the repression caused by elevated levels of guanosine tetraphosphate (ppGpp). During starvation conditions, the alarmone ppGpp is synthesized. Overexpression of ppGpp causes inhibition of dnaAp2 promoter transcription, which is alleviated when the GC-rich DS is mutated to an AT-rich sequence. We conclude that this site is responsible for mediating the stringent control of the dnaAp2 promoter. Incorrect timing of initiation in the cell cycle results in asynchronous replication. Strains with a mutated DS, DnaA box, or Fis binding site in the dnaA promoter region on the chromosome have increased levels of dnaA gene expression. These mutants initiate DNA replication synchronously in minimal M9 medium as shown by flow cytometric assays. In rich medium, the DS, mutant showed very limited asynchrony, whereas the other mutants replicated synchronously.
