Gene Regulation/Response to Environment Flashcards
E. coli has 6 NAPs. Which NAP undergoes oligerimization to bridge between adjacent segments of DNA?
H-NS
E. coli has 6 NAPs. Which NAPs induce severe bends in DNA?
Fis & IHF
E. coli has 6 NAPs. Which highly conserved NAP acts like a histone?
HU
E. coli has 6 NAPs. Which NAPs condense DNA under stress conditions?
Dps & CbpA
Describe how Topoisomerase II induces a negative supercoil.
- GyrB binds DNA.
- GyrA induces a DSB, remaining covalently bound to each end.
- GyrA passes the intact strand through the DSB using ATP hydrolysis.
- GyrB re-ligates the DSB.
Which antibiotic targets & stabilises the Gyrase covalent complex.
Quinolone
How does Topoisomerase I relax a negative supercoil?
- Topo I induces a SSB.
- The intact strand is passed through the break.
- The SSB is re-ligated.
What is a Shine-Dalgarno sequence?
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.
Describe the ideal bacterial promoters.
Two sequences, -35 (TGTTGACA) & -10 (TATAAT) with a 17bp spacing.
What regulates the suitability of a promoter sequence?
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.
Describe Rho-dependent trancription termination.
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.
Describe Rho-independent transcription termination.
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.
What is σ70
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
What is σ38
General Stress, 1/3 abundance of σ70. Complex regulation to ensure only activated in stress conditions. Same promoters as σ70
What is σ32
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
What is σ28
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
What are σ24, σ19, σ54
σ24/σE Extracytoplasmic Stress, σ19/σJ Ferric Citrate transport, σ54/σN Nitrogen-related.
How are σ factors regulated
Secondary Structure of Transcript, Proteolysis, Anti-σ factors, Anti-anti σ factors
What is a two-component system?
TCS allow bacteria to sense and respond to their environment. Typically consists of a ‘sensor’ histidine kinase, and an ‘effector’ response regulator.
Describe simple TCS mechanism.
- 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.
Give some examples of histidine kinase domain variation.
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)
Give some examples of Response Regulator domain variation.
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
What are the functions of TCS accessory proteins?
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
How does RR activation occur in RstA?
- RstB phosphorylates RstA, triggering autodimerisation
- DBD changes conformation & searches for suitable binding site
- DBD bind in adjacent major grooves (requires adjacent binding sites)
- RR bound to DNA makes contact with the RNAP α-subunit c-terminal or σ-factor to activate transcription
