Bacterial Cell Cycle and ColE1

Question 1

how is septum formation associated with doubling time of bacteria?

Answer 1

• a septum is the division in the middle of the mother cell to get a separation and formation of the two resultant daughter cells
• it will always take 20 minutes to form the septum and split the cell into 2

Question 2

What are the values (minutes) of C and D, and what do C and D stand for?

Answer 2

• It requires 40 minutes to replicate the bacterial chromosome (at normal temperature) known as C
• Completion of replication cycle triggers a bacterial division 20 minutes later known as D
• C+D=60 minutes; this is the total time between the initiation of DNA replication and cell division
• The doubling time of e. Coli can vary over a 10x range, depending on growth conditions

Question 3

What is a unit cell?

Answer 3

an entity 1.7 micrometers long; one origin per unit cell.

Question 4

What is the significance of 1.7 microns in terms of the number of origins present in E. coli?

Answer 4

• A rapidly growing cell with two origins will be 1.7-3.4 micrometers long
• At 10 minutes after division, the cell mass increases sufficiently to support an initiation at both origins
• 1.7 microns is used to Measure its length to see if its replicating
• Up to 1.7 microns long - then there is only one origin working - longer than this, it started replication for the daughter cells
• Anything between that will have 2 origins going at the same time
• Keep doubling to see how many origins there will be

Question 5

How could you experimentally change the copy number of the ColE1 plasmid?

Answer 5

• Change the activity level of the promoter you can alter the copy number
• Not making as much of a negative regulator so it takes longer to shut down replication
• End up with a higher copy number
• Made the promoter that makes the primer strong, you can also increase the final copy number through this mechanism as well

Question 6

how does rna 1 negative regulator “count” the copy number?

Answer 6

• P-1 promoter regulates the expression level (abundance) of RNA 1 regulatory transcript
• Alter the promoter, alter the copy number

Question 7

How does the cell know when to initiate the replication cycle?

Answer 7

• Rapidly growing cells are larger and posses a greater number of origins (multi forked chromosomes)
• Initiation occurs at a constant ratio of cell mass to the number of chromosome origins

Question 8

How is cell mass titrated?

Answer 8

there are two competing models: the current and alternative model

Question 9

current model for mass titration

Answer 9

Current model suggests that initiation is controlled by the accumulation of a positive acting factor. Accumulation of a critical amount would trigger initiation (this factor is dilute in newly divided cells). DNA A protein is likely the candidate

Question 10

alternative model for mass titration

Answer 10

Alternative model suggests that initiation is controlled by the accumulation of a negative acting factor (an inhibitor) inhibitor may be synthesized to a fixed level (no initiation) and diluted below at an effective level in larger cells (initiation)

Question 11

How many replication origins are active in an e. Coli cell that is 2.1 micrometers in size?

Answer 11

2

Question 12

Xer

Answer 12

• Site specific recombination system to resolve a dimeric genome into monomer genomes after replication
• Enzyme that makes the cut that resolves the two genomes
• Resolvase

Question 13

Dif

Answer 13

location where Xer makes the cut

Question 14

how fast does the replication fork move in bacterial replication in the cell cycle?

Answer 14

50,000 bp/min

Question 15

multifork chromosome

Answer 15

• Fast rate of growth
• The new strands have an active origin and another pair of replication fork forms

Question 16

doubling time

Answer 16

• time needed for cell numbers to double.
• In e. Coli they range from 18 minutes to slower than 180 minutes.
• Average in lab in 60 minutes

Question 17

explain how the doubling time for E. coli can be as little as 18 minutes, yet, it always takes 40 minutes to replicate the DNA and another 20 minutes to prepare the cell to divide.

Answer 17

• If doubling time is less than 60 minutes, a replication cycle is initiated before the division resulting from the previous replication cycle
• Fast rates of growth therefore produce multi forked chromosomes
• There is a fixed interval of 60 minutes between initiation of replication and cell division
• At 10 minutes, initiation occurs at both origins on the partially replicated chromosome
• There is an overlap of replication cycles
• A replication cycle is started before the original replication cycle has a change to divide
• Start replicating the next generation before you are done with the first generation
• The doubling time is so much faster because once your first generation has divided, you already have many more of the next generations that were already forming and replication that are ready for cell division

Question 18

how do you separate daughter genomes?

Answer 18

• the oriC has an affinity for the membrane at a specific site and the termination site has an affinity at the septum
• the bacterium is physically separating the genomes
• steps: the origin and terminus are located mid cell in newborn cells, newly replicated origins move apart, cell septum divides, and origins and termini reorient and the cell divides

Question 19

why is it necessary to separate genomes in dividing cells?

Answer 19

• to ensure a copy ends up in each cell
• to prevent recombination machinery from being active

Question 20

what happens when recombination occurs in a dividing cell?

Answer 20

• when the cells experience recombination, they are stuck and cannot seperate into their own cells to divide
• if recombination occurs, a genome dimer is formed that must be resolved by a site-specific recombination - basically a second recombination event
• the xer-site specific recombination system resolves a dimeric genome into monomeric units
• the xer-directed recombination occurs at the dif site which must be within 30 kb from the septum that is forming between daughter cells
• xer is the recombinase and dif is where xer acts

Question 21

how is bacterial replication is connected to the cell cycle?

Answer 21

• Frequency of initiation adjusted to fit rate the cell is growing
• The completion of a replication cycle triggers to cell division

Question 22

how many levels of regulation are there is the ColE1 plasmid?

Answer 22

3

Question 23

what is an example of positive regulation in ColE1?

Answer 23

the RNA 2 primer enhances replication by providing a 3” OH end

Question 24

what are the examples of negative regulation in ColE1?

Answer 24

• RNA 1 repressor shuts down replication but allows continued transcript of RNA 2
• Rom protein helps to shut down replication because it enhances RNA2/RNA1 duplex formation

Question 25

RNA 1

Answer 25

negative anti-sense regulator for RNA 2 that forms a hairpin structure to destabilize the RNA

Question 26

RNA 2

Answer 26

a primer for DNA polymerase, forms a hairpin structure to stabilize the sequence

Question 27

Rom protein

Answer 27

negative regulator that works in conjunction with RNA 1 to destabilize RNA 2

Question 28

Explain the process of ColE1 replication and explain how it is regulated.

Answer 28

• starts with initiation through transcription
• pColE1 is a primer that controls the rate of DNA initiation
• transcription must pass through the origin
• RNAase H cuts the transcript as it passes through the origin
• RNA 2 (the primer) forms a hairpin structure at the 5’ end to stabilize the RNA in order for RNAase H to cut and DNA polymerase to bind
• after the end is processed, DNA polymerase can replicate
• however RNA 1 also forms a hairpin structure that is antisense to RNA 2
• this effect straightens the two hairpin structures and destabilizes it so RNAase H cannot cut an dDNA pol cannot bind
• another negative factor called Rom protein enhances the destruction of hairpins by ensuring that they bind