Lec 10 - Genome Structure

Question 1

How are transcription units orientated along the chromosome and what is the reason for this?

Answer 1

Orientated in same direction that DNA replication occurs to avoid DNA pol and RNA pol clashes

Question 2

What is the Nucleoid?

Answer 2

Non membrane bound
Region of chromosome where dsDNA is located
Around 50 domains make up the nucleoid - each loop associated with nucleoid associated proteins and each loop is coiled

Question 3

Describe +ve and -ve supercoils.

Answer 3

+ve = winding > frequently
overwinding
-ve = winding < frequently
underwinding

Question 4

Describe the enzymes that regulate supercoiling.

Answer 4

INTRODUCING NEGATIVE SUPERCOILS - Gyrase
GyrB binds to DNA
GyrA introduces DSB in DNA
2 ends remain intact through interactions with GyrB
GyrA ATPase activity passes unbroken strand through the DSB and GyrB rejoins the nicked strand
GyrB releases the unbroken strand

REMOVING NEGATIVE SUPERCOILS - Topoisomerase
Topo I binds to DNA strand and nicks one strand of the helix
Topo I passes the unbroken strand back through DSB and removes 1 supercoil
Nicked strand is religated

Question 5

Which type of antibiotics target Type II toposisomerases and how do they work?

Answer 5

Quinolone antibiotics

Cause formation of covalent complex between Gyr and DNA therefore preventing DNA replication

Question 6

Describe the 4 different types of NAPs and give examples.

Answer 6

HU - most conserved NAP. involved in the condensation of DNA into fibres
IHF- introduces drastic bends into the DNA. may be involved in regulating gene activity
H-NS - bridges separate sections of DNA
DPS - protects stationary phase DNA from attacks by ROS/UV

Question 7

What are the 2 types of NAP that remains fairly constant throughout bacterial life cycles?

Answer 7

HU and H-NS

Question 8

How is the E. coli chromosome organised?

Answer 8

6 macro domains
each macro domain has 1mil bp
SOLENOID form:
each macro has 10 helices (100,000bp each)
each helix has 10-12 micro domains w/ H-NS proteins
further supercoiling in each loop to reduce the diameter
Molecular packing - Mg2+ and +vely charged NH3 polyamines bind to the P backbone and neutralise the -ve charge to reduce electrostatic repulsion

Question 9

Describe Rho dependent and independent termination.

Answer 9

DEPENDENT:
Rho binds to GC rich region (after translational stop)
Rho uses its ATP-dependent helicase activity to reach the RNAP
once contact made, transcription stops

INDEPENDENT:
GC hairpin loop on mRNA forms and once this structure interacts w/ RNAP transcription stops