BACTERIAL GENOMES

Question 1

Describe the genome of E.coli

Answer 1

It is supercoiled

Question 2

What mediates the genome of E.coli?

Answer 2

Adding or removing turns causes tension.
1 Topoisomerase relaxes SC via a scissor action.
2 Topoisomerase breaks both strands
DNA gyrase causes negative supercoiling

Question 3

What is a multipartite genome?

Answer 3

Genome has been divided two or more times

Question 4

Describe DNA replication in E.coli

Answer 4

E.coli has a single origin of replication on it’s chromosome.
Specialised Helicase recognises and bind to site, opens up DNA.
Two Y-shaped structures called replication forks are formed, together making up what’s called a replication bubble. The replication forks will move in opposite directions as replication proceeds.
Single Stranded Binding proteins coat the region around the RF to prevent rewinding.
Topoisomerase works at region ahead of RF to prevent SC.
Primers make RNA primers that are complementary to DNA. These bind and Pol II can extend them in the 3’ direction. This is the leading strand.
Other new strand is 5’ to 3’ is tricky as DNA runs away from fork, lagging strand needs new primer for every Okasaki fragment.
DNA Pol 1 removes RNA primers and replaces with DNA primers.
DNA ligase fills in nicks when DNA primers leave.

Question 5

What is a replicon and what happens to it during cell cycle?

Answer 5

A replicon is a region that initiates and completes synthesis. It must be duplicated at least once so every daughter cell has copy.

Question 6

What type of replication does E.coli undergo?

Answer 6

Bidirectional replication

Question 7

OriC’s role in E.coli’s (prokaryotes) DNA replication

Answer 7

oriC binds to DNA and distorts it causing complex to close.
DNA B and helicase bind with help of DNA C which is released. This opens DNA. DNA B unwinds DNA with SSB and DNA gyrase. DNA G acts as primer for replication.

Question 8

What controls E.coli’s replication?

Answer 8

Dam-methylase. GATC motifs are methylated by Dam-methylase at the oriC.

Question 9

Describe initiation in E.coli

Answer 9

Replication happens when all 14 copies of GATC are methylated.
So only one copy is made so new DNA is hemi-methylated, needs to be remethylated for replication.

Question 10

Describe termination in E.coli

Answer 10

Two RF meet in opposite directions known as the terminus region. TR stops fork progression.

Question 11

Describe the Meselon-Radding model

Answer 11

One strand cleaved by endonuclease.
Produces a chain that sticks out.
This chain invades homologous DS DNA.
RecA catalyses.
Ligation causes creation of Holiday Junctions.
HJ can undergo branch migration. 
RuvC cleaves junction.

Question 12

Describe DS homologous recombination in E.coli

Answer 12

Rec BCD binds to end of dsDNA and unwinds it using helicase.

Question 13

Describe transposition in Salmonella

Answer 13

Transposition = Inverting a sequence which can lead to gene expression.
In Salmonella, the Hin region encodes for invertase.
H1 flagella protein is separated from Hin, and has its own promoter.
H2 encodes for repressor of H1 in operon with repression gene. The promoter for H2-rep is in Hin region.

Question 14

When are the two primer pairs expressed?

Answer 14

P1 H1 is expressed because Hin is in the wrong direction.

P2 H2 expressed when Hin is flipped because promoter is in the right orientation.