Lecture 2 - Regulation in bacteria Flashcards
How does replication initiation take place in E. coli?
DnaA recognises binding sites within oriC
DnaA binding and oligomerisation
Unwinding induced at DUE
Helicase loader DnaC facilitates binding of DnaB on ssDNA
Helicase recruits primase and clamp (DnaG and DnaN)
Describe how DnaA is converted between the active and inactive forms
DnaA exists in 3 different forms: DnaA on its own, a version bound to ATP and one bound to ADP. Only the ATP version is active - once it has lost a phosphate in ATP hydrolysis and become the ADP-bound form it is inactive. There is a protein called Hda that stimulates ATP hydrolysis and so converts it from active to inactive form. Hda combines to DNA and it can also combine to DnaN (the beta clamp). The beta clamp is only present when initiation has passed and the elongation phase has started. Hda binds via clamp-binding domain. Once this 3 protein complex has formed Hda can convert DnaA into the inactive form. This process is called RIDA. It is regulatory because it only happens at a certain phase of DNA replication. So - DnaA binds, origin fires, replication fork is assembled and then Hda can inactivate.
Describe how transcription of DnaA is autoregulated
DnaA inhibits its own transcription- can do this because it has DnaA boxes in its promoter. Transcription happens, making the DnaA protein. Once that protein has been involved in replication it loses its phosphate and becomes ADP-bound. This form can then bind the promoters of its own gene which represses its own production. The ATP-bound version is much stronger at repressing. This feedback mechanism makes sure there is never too much DnaA in the cell because then it will repress its transcription and not enough will be made.
Describe how methylation takes place in bacteria
Methylation is addition of a methyl group to a particular carbon. Bacterial DNA is methylated at As and Cs if they are in GATC motif. All bacterial DNA is methylated on both strands, but when replication takes place the new DNA won’t have had a chance to be methylated.
Hemimethylated DNA resistant to initiation because of protein called SeqA which binds to hemimethylated DNA and prevents DnaA from forming its oligomer that you need for more initiation. This prevention of initiation is called sequestration.
SeqA binds hemi-methylated GATCimmediately after initiation
SeqA blocks DnaA oligomer formation and prevents immediate re-initiation “sequestration”
Bound SeqA persists for a third of cell cycle and continues to recruit more SeqA
SeqA dissociation and methylation of GATC by Dam methyltransferase relieves the sequestration of oriC
dnaAtranscription is repressed in a SeqA-dependent manner
Give an example of positive regulator of replication
IHF = Integration Host Factor
IHF is a sequence-specific DNA-binding protein that bends DNA
The bend brings adjacent DnaA-boxes into close proximity and facilitates extension of the DnaA oligomer
IHF binding is cell-cycle regulated and independent of DNA replication
Give an example of a negative regulator of replication
datA promotes hydrolysis of DnaA-ATP once IHF has bound
DARS1 and DARS2 promote conversion of DnaA-ADP to DnaA-ATP
datA is negative regulator of DNA replication
IHF changes the shape and then datA shifts the balance towards the inactive form of DnaA
DARS1 and DARS2- positive regulators
If there is more active form in the cell, there is increased likelihood of oriC firing
Describe the negative regulation of DNA replication
Inhibition of DnaA activity via -Transcription control -RIDA -datA Titration of DnaA Sequestration oforiC seqA binding to hemi-methylated newly replicated DNA Fis inhibits DNA unwinding at oriC by blocking DnaA and IHF binding
Describe the positive regulation of DNA replication
DARS1 and DARS2 bind DnaA-ADP and promote regeneration of DnaA-ATP
Methylation of GATC by Dam methyltransferase relieves oriC sequestration
IHF binding induces a bend in DNAand helps oligomerisation
Stimulation of DnaA-ATP oligomerisation by DiaA
How do bacteria replicate so quickly?
Origins fire bidirectionally
Multiple replication forks and rounds of replication
How does cell division take place?
Segregation is continuous and concurrent with DNA replication
The core of the division machinery is the Z-ring
Polymers of FtsZ form the Z-ring
Steps of cell division
Z-ring assembled on the membrane at the mid-cell position. This step is under spatial and temporal control to ensure that the Z-ring is assembled between segregated chromosomes
Remaining cell division proteins added to form the divisome
Divisome activated to synthesize septal peptidoglycan. Cell wall degrading enzymes only activated after septal cell wall synthesis has begun.
Z-ring formation occurs before septation
Cells can begin septation when 70% of the chromosome has been replicated
Blocking initiation affects Z-ring positioning
The earlier the initiation block, the less likely a Z-ring forms
Completion of initiation allows mid-cell Z-rings to form at wild-type level
Therefore the early stages of DNA replication
must trigger the first stage of cell division.
As replication nears completion, the division machinery accumulates and assembles at the division site in preparation for septation.
Division does not occur until the mid-cell has been cleared of DNA.
What is filamentation?
Rod-shaped cells replicate but do not divide
Septa do not form
Stress response e.g. DNA damage, antibiotics
Can slow down the uptake of bacteria by immune cellsand promote survival within host tissues
What is endoreduplication?
Eukaryotic cells skipping division phase
Occurs in many tissues e.g.endosperm of developing seeds
Drosophila salivary glands
Endocycle = alternating replication and gap phases
Heterochromatin and specific euchromatic regions are under-replicated
