Gene Regulation/Response to Environment

Question 1

E. coli has 6 NAPs. Which NAP undergoes oligerimization to bridge between adjacent segments of DNA?

Answer 1

H-NS

Question 2

E. coli has 6 NAPs. Which NAPs induce severe bends in DNA?

Answer 2

Fis & IHF

Question 3

E. coli has 6 NAPs. Which highly conserved NAP acts like a histone?

Answer 3

HU

Question 4

E. coli has 6 NAPs. Which NAPs condense DNA under stress conditions?

Answer 4

Dps & CbpA

Question 5

Describe how Topoisomerase II induces a negative supercoil.

Answer 5

1. GyrB binds DNA.
2. GyrA induces a DSB, remaining covalently bound to each end.
3. GyrA passes the intact strand through the DSB using ATP hydrolysis.
4. GyrB re-ligates the DSB.

Question 6

Which antibiotic targets & stabilises the Gyrase covalent complex.

Answer 6

Quinolone

Question 7

How does Topoisomerase I relax a negative supercoil?

Answer 7

1. Topo I induces a SSB.
2. The intact strand is passed through the break.
3. The SSB is re-ligated.

Question 8

What is a Shine-Dalgarno sequence?

Answer 8

An SD sequence is typically found before the start codon (ATG) in protein coding transcript. It’s typically 6-8 nt in length (AGGAGG). It is thought to help regulate translation.

Question 9

Describe the ideal bacterial promoters.

Answer 9

Two sequences, -35 (TGTTGACA) & -10 (TATAAT) with a 17bp spacing.

Question 10

What regulates the suitability of a promoter sequence?

Answer 10

Whether the two promoter sequences are on the same face of DNA; the level of supercoiling. Whether it’s in open chromatin, or containing any secondary structures that disrupt RNAP binding.

Question 11

Describe Rho-dependent trancription termination.

Answer 11

Multiple copies of Rho bind to a transcription termination sequence, they wrap mRNA around themselves faster than the rate of transcription. Contact with the polymerase acts as a stop signal.

Question 12

Describe Rho-independent transcription termination.

Answer 12

RNA super structure forms due to a GC-rich termination sequence. GC rich stem-loop forms spontaneously, contact with the polymerase acts as a stop signal.

Question 13

What is σ70

Answer 13

House keeping genes, highest abundance during exponential growth, 2 anti-σ70 factors: 1. Rsd, binds to RNAP core, lowers affinity for σ70 to that of σ38. 2. HscC acts as DnaK chaperone, binds and sequesters σ70 for deg. Binds to promoter elements -35 TGGACA and -10 TATAAT

Question 14

What is σ38

Answer 14

General Stress, 1/3 abundance of σ70. Complex regulation to ensure only activated in stress conditions. Same promoters as σ70

Question 15

What is σ32

Answer 15

Heat Shock, encoded by rpoH, has a high level of secondary structure under normal temp, cant be translated and is degraded (controlled by σ70 so mRNA is always present).
High Temp melts secondary structure and normal degradative pathways are overloaded. Is efficiently translated and allows expression of σ32 controlled genes including different lipid groups, chaperones and leads to upregulation of degradation pathways

Question 16

What is σ28

Answer 16

Motility and Flagella. 1/2 abundance of σ70, lower under heat shock. Promoter has extended -10 element GCCGATAA, GC Region is key.
1. σ70 controls class 2 operon, encodes fliA (σ28) and proteins that make up the base of the flagella
2. σ28 controls class 3 operon, contains fliC, encodes protein in filament of flagella.
3. FlgM, an anti-σ28 factor is secreted from the cell until the hook of the flagella as been synthesized

Question 17

What are σ24, σ19, σ54

Answer 17

σ24/σE Extracytoplasmic Stress, σ19/σJ Ferric Citrate transport, σ54/σN Nitrogen-related.

Question 18

How are σ factors regulated

Answer 18

Secondary Structure of Transcript, Proteolysis, Anti-σ factors, Anti-anti σ factors

Question 19

What is a two-component system?

Answer 19

TCS allow bacteria to sense and respond to their environment. Typically consists of a ‘sensor’ histidine kinase, and an ‘effector’ response regulator.

Question 20

Describe simple TCS mechanism.

Answer 20

1. Membrane-bound histidine kinase receives signal. 2. Catalytic ATPase domain binds ATP and phosphorylates a conserved histidine in the transmitter domain. 3. Phosphorylated histidine kinase transiently interacts with it’s cognate RR. 4. Phosphoryl group is transferred to the receiver domain on the RR. 5. Effector domain on the RR is now active.

Question 21

Give some examples of histidine kinase domain variation.

Answer 21

Typically they have TM domains to anchor them to the membrane, a catalytic ATPase domain and a transmitter domain. Additional domains that can be found are: Pas or GAF domains which bind small ligands. HAMP domains can transmit conformational changes along the protein to the CA domain. (4 helices which rotate against each other)

Question 22

Give some examples of Response Regulator domain variation.

Answer 22

All RRs have a receiver domain (RD), RD’s found on their own have unknown functions.
a) DNA-binding domain (DBD)
b) AAA+ domain, AAA+ ATPase activity allows the activation of σ54 controlled genes
c) GGDEF domain, involved in c-di-GMP synthesis key in the sessile-motile switch.
d) Methyltransferase domains key in chemotactic responses

Question 23

What are the functions of TCS accessory proteins?

Answer 23

a) Separate sensory proteins can transmit signals to the HK
b) Scaffold proteins can facilitate transmission between HK and RRs
c) Connector proteins can swap signals between TCS allowing system cooperativity
d) Allostery; RR without an output can feed into a different TCS

Question 24

How does RR activation occur in RstA?

Answer 24

1. RstB phosphorylates RstA, triggering autodimerisation
2. DBD changes conformation & searches for suitable binding site
3. DBD bind in adjacent major grooves (requires adjacent binding sites)
4. RR bound to DNA makes contact with the RNAP α-subunit c-terminal or σ-factor to activate transcription

Question 25

Describe the acid response in E Coli

Answer 25

1. EvgSA senses low pH, induces expression of safA and ydeO
2. SafA, a membrane-bound connector protein, activates PhoQ
3. PhoQ’s RR PhoP regulates the ira gene iraM
4. IraM binds RssB preventing σ38 degradation
5. gadE can now be transcribed; requires σ38 and YdeO
6. GadE stimulates acid response

Question 26

How does evolution and fidelity relate to TCS?

Answer 26

HKs maintain phosphotransfer specificity to their RRs. Duplication of TCS genes can create two pathways that crosstalk at the level of phosphotransfer. Accumulation of specificity-determining residues over time insulates the pathways against crosstalk.
HK-RR specificity can be switched by mutating just a few specificity-determining residues. E.g. RstB’s phosphotransfer specificity can be switched to that of EnvZ by mutating just 3 residues.