1
Q
define a replicon.
A
A DNA unit capable of Replication and Inheritance
2
Q
who discovered replicons?
A
Jacob, Brenner & Cuzin, 1963
3
Q
give 2 examples of replicons
A
chromosomes and plasmids
4
Q
4 facts of chromosomes
A
– Essential for viability
– Generally 1; sometimes >1 (Vibrio = 2)
– Sizes 0.5 - 5Mb (500-5000 genes); some ~30MB
– Mostly ccc DNA ; some linear DNA (Borrelia)
5
Q
3 characteristics of plasmids
A
– Super-numerary, non-essential for viability
– May confer new phenotype (eg. resistance, catabolism)
– Smaller than chromosomes: 1 – 200kb
6
Q
how is replication controlled
A
ori usage and copy number
7
Q
who discovered Isolated Nucleoids?
A
Pettijohn (1971)
8
Q
what do nucleoids contain?
A
DNA, RNA, Proteins
– Major proteins; RNA Polymerase DNA Gyrase
9
Q
what was pettijohns experiment?
A
- Treatment with detergent & protease
- Releases anchored loops of DNA
- DNA in loops is supercoiled
- loops isolated & anchored at base
10
Q
who discovered the Folded bacterial Chromosome?
A
Worcel & Burgi, 1972
11
Q
what are the 4 Chromosomal Macrodomains?
A
Ori, Left, Right, Ter
12
Q
name 3 Domain-specific binding proteins?
A
– SeqAp
– SlmAp
– MatPp
13
Q
what does SeqAp do?
A
- binds Ori, R, L localises oriC
- Negatively regulates timing of replication + initiation.
14
Q
SlmAp do?
A
- binds Ori, R, L
- chromosome positioning
15
Q
what does MatPp do?
A
- binds Ter and localises/anchors terC
- Stops premature chromosome segregation
16
Q
types of DNA compaction in Nucleoid
A
- Long range loop domain folded chromosome x25 compaction
* Short range supercoiling condensed nucleoid x860 compaction.
17
Q
DNA concentration in nucleiod?
A
- 2-20mg/ml DNA
- 80% mass nucleoid, only 5% vol
18
Q
what are the Structural proteins of the Nucleoid?
A
- Nucleoid Associated Proteins (NAPs)
- SMC (chromosome organising) proteins
19
Q
name 3 NAPS
A
HU, IHF, H-NS
20
Q
whats HU?
A
- 9.5kDa NAP
- binds structural distortions/stabilises bends
- binds & constrains supercoils
21
Q
whats IHF?
A
- sequence-specific
- bends DNA
22
Q
what does H-NS do?
A
- binds AT rich bent DNA
- forms bridge between two DNAs
- DNA compaction.
23
Q
what are NAPS?
A
- small DNA binding proteins
- abundant
- basic
- dimeric
- highly conserved
- nonessential
24
Q
what is the structure of SMC proteins?
A
– Large V-shaped.
– Two DNA-binding domains connected by long coiled region with flexible hinge
25
how does MukBEF act as a Condensin?
- Nucleoid compaction & organisation
| - hinge and arms and ATP.
26
how do NAPs affect gene expression?
* Interactions with DNA affecting shape
* bind AT-rich promoters
* Sets expression state of genes
27
whats the most stable form of bacterial DNA?
360bp covalently closed circle of B-Form DNA
28
what is b-form DNA?
- Right-handed helix
- 10.5bp/helix turn
- Bases lie flat & perpendicular to helix axis
29
how does b form supercoil?
- breaks 1 strand + Unwinds 4 helix turns and reseals break
| - DNA changes shape to its most stable state lowest free energy
30
what are the Effects of EthBr intercalation into DNA?
-unwinds negative supercoil to introduce a positive sc
31
whats the Action of E.coli Topoisomerase 1 on DNA?
- type-I topoisomerase Breaks 1 DNA strand, holds ends, passes 1 (other) strand through, reseals nick.
- introduces one positive SC
- releases one negative SC
32
Action of E.coli DNA Gyrase?
- A type-II topoisomerase
- Breaks 2 strands of DNA, passes duplex through, reseals break
- introduces two negative SCs
33
How are the linked circles decatenated?
Type II Topoisomerase
| In E.coli, TopoIV
34
how do fast growing cells differ from slow?
larger with more DNA than slow cells
35
how does Origin/Terminus ratio change with growth rate?
- Fewer Ter/Ori at faster growth rates
| - dosage of genes near Ter falls at fast growth rates
36
describe Replication of the E.coli Chromosome?
1. 2 Rep Forks each traversing half chromosome
2. Cell division occurs constant time after Termination
3. 1 replication fork polymerises 2.3Mb in 40 minutes
37
how does Rate of Cell Growth regulate frequency of initiation?
1. Initiator Accumulation- Initiator made proportional to growth rate accumulate to critical level for initiation Initiator consumed at initiation
2. Initiator Dilution- Inhibitor produced at initiation Diluted by cell growth (volume increase) Initiation when concentration falls below threshold
38
where is E.coli's Initiation?
oriC
39
describe oriC?
1. Unique origin of bidirectional replication
2. Isolated DNA replicates freely as minichromosome
3. Minimal sequence 245bp
– 5 x DnaA-ATP binding sites
– 3 x AT-rich 13mer sequences
– binding sites for IHF, FIS, HNS
40
what is DnaA?
1. 52kDa protein monomer
2. binds ATP hydrolysed to ADP during replica’s
3. DnaA-ATP binds dnaA boxes in oriC, PdnaA, datA
4. DnaA-ATP opens oriC at AT rich repeats
41
how is DnaA expression controlled?
auto-regulation via dnaA box in promoter limits amount of protein made
42
describe Initiation at oriC.
1. DNA in correct topology negatively sc
2. binds DnaA protein which Binds ATP
3. DnaA-ATP binds to dnaA boxes in oriC Wraps oriC DNA around multimer of DnaA-ATP
4. Opens oriC at A T rich region
43
how does replication proceed at initiation at oriC?
1. DnaB/C helices complex loads, unwinds DNA
2. DnaG primas enters, synthesises primer
3. Pol III complex with DnaN sliding clamp loaded
4. DnaA-ATP hydrolysed to ADP
44
what are the negative controllers of OriC?
1. SeqA - Sequestering protein
2. idaB- Replication Inhibition of DnaAp
3. datA- DnaA titration locus
45
how does SeqA work?
sequesters hemi-methylated oriC and PdnaA to membrane
46
how does idaB work?
– hydrolyses ATP to ADP inactivates DnaAp
47
how does datA work?
– depletes cell of free DnaA-ATP protein
48
describe Initiation control by sequestration of oriC by seqA
1. oriC becomes hemi-methylated by initiation
2. seqA protein binds to hemi- methylated DNA
3. sequesters to membrane
4. methylation of other strand by dam blocks seqA binding
49
how is dnaA activity controlled by ATPase action of idaB of the Sliding Clamp?
1. idaB hydrolyses ATP on dnaA to ADP
| 2. dnaA-ADP unable to initiate replication
50
whats the net result of OriC control cycle?
All active DnaA removed oriC unaccessible
51
how is the oriC cycle switched on?
1. oriC free, fully methylated, supercoiled
2. DnaA-ATP accumulates to critical concentration autoregulation of dnaA gene.
3. DnaA-ATP binds to oriC initiation triggered
4. oriC replicated converted to hemi-methylated state
52
how is OriC switched off?
1. Hemi-methyl’d oriC sequestered to membrane by SeqA
2. oriC unavailable
3. beta-clamp hydrolyses ATP to ADP; DnaAp bound at oriC inactivated
4. dnaA gene replicated GATCs hemi-methylated & sequestered,DnaA synthesis stops released & activated after delay
5. datA locus replicated binds any free DnaA-ATP
53
how is danA switched off at Initiation at oriC?
1. idaB hydrolyses ATP dnaA-ADP inactive for initiation
2. hemi-methylated oriC sequestered by seA
3. excess dnaA removed by datA-binding
54
whats The Chromosomal End Game?
1. Termination of replication
2. Resolution of multimers to monomers
3. Decatenation of linked products
4. Segregation to daughter cells at division
55
describe Cell Division of E.coli?
1. FtsZ polymerises into Z-ring at mid-cell needs GTP hydrolysis
2. MinCDE proteins control position of Z-ring
– MinCD oscillates from pole to pole every ~30sec establishes conc gradient; low at middle high at poles
3. Other proteins attach to Z-ring Septal Ring
4. Z-ring septal ring contracts inwards pulling in membrane and wall
5. Nucleoid occlusion stops Z-ring complete contraction and closure
6. FtsZ controls TopIV decatenation and Xer/difresolution of multimer products of replication.
56
whats the Movement of Nucleoid at chromosome segregation?
1. oriC foci at 1⁄4 and 3⁄4 positions SeqA moves oriC
2. Pol III replic’n complex at mid cell – Spools DNA through
3. terC at mid cell
4. MukFEB complex (cohesin/condensin) moves replicated chromosomes apart
57
what do MinCD proteins do?
determine site of cell division septum by oscillating every 30 sec inhibiting FtsZ polymerisation
58
example of an SMC protein
MukBEF
59
what do SMC proteins do?
- compaction
- cohesion
- loop domain- organisation,
- movement/segregation of nucleoid
Microbial Genomics
flashcards
Decks in class (7)
# Cards
