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Microbiology 2 > Prokaryotic gene regulation > Flashcards

Prokaryotic gene regulation Flashcards

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Biology 101 flashcards
1
Q

what are bacteria?

A

free-living organisms that need to quickly adapt to changing environmental conditions to survive:
- often live in competitive environments so must maximise efficiency

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2
Q

what is the typical structure of a bacterial genome?

A
  • single circular chromosome
  • DNA is densely coding: few introns, no spliceosome and no repeating sequences of junk DNA
  • mostly encodes protein or functional RNAs
  • short intergenic distances (60-70bps) - promoters
  • operons are common and some are monocistronic - multiple genes transcribed in a single transcription unit
  • transcription units orientated in same direction as chromosome replication to avoid polymerases crashing
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3
Q

how is the nucleoid organised in bacteria?

A
  • constrained into domains
  • accessible to DNA and RNA polymerases so highly dynamic
  • DNA is highly coiled
  • coiling is independent between loops, so each can be further compressed or relaxed as needed
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4
Q

what are the features of the E. coli nucleoid?

A
  • 4.6mbp genome
  • a circle of DNA 1.6mm long inside a 2um cell
  • approx 400 domains in E. coli
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5
Q

what are nucleoid-associated proteins (NAPs)?

A
  • analogous to chromatin proteins in eukaryotes
  • they bind to DNA to help organise it and take up less space in cell
  • differ in function and affect on DNA

E. coli have 6 NAPs

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6
Q

What are the 6 NAP types in E. coli? what are their roles?

A
  1. H-NS
  2. Fis
  3. IHF
  4. HU
  5. Dps
  6. CbpA
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7
Q

what is the role of H-NS NAP?

A

bridges adjacent segments of DNA

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8
Q

what is the role of the Fis NAP?

A

Induces sharp bends

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9
Q

what is the role of the IHF NAP?

A

induces sharp bends

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10
Q

what is the role of the HU NAP?

A

condenses DNA into a fibre
- the most conserved NAP

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11
Q

what is the role of the Dps NAP?

A

condenses DNA to protect it from damage during stress, expressed in stationary phase

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12
Q

what is the role of the CbpA NAP?

A

condenses DNA to protect it from damage during stress, expressed in stationary phase

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13
Q

why is DNA supercoiling useful?

A

uncoiled/relaxed DNA = coils approx 10bp per turn
- supercoiling enables compaction of DNA to fit into the cell
- as more twists are added into the DNA, the structure becomes more compact and energy is held within the knot
- supercoiling can add/remove energy that can be used for transcription

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14
Q

what are the types of DNA supercoils?

A

positive supercoil: over-twisting the DNA helix
negative supercoil: under-twisting the DNA helix

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15
Q

what controls DNA supercoiling?

A

topoisomerases

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16
Q

how many topoisomerases does E. coli have?

A

I-IV
- Topo I, III and IV introduce positive supercoils
- Topo II (gyrase) forms negative supercoils

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17
Q

how does Topo II (gyrase) form negative supercoils?

A

GyrB/GyrA complex:
- GyrB binds DNA and GyrA makes a double-strand break, remaining covalently bound at each end
- GyrA (ATPase) hydrolyses ATP which causes conformational change that passes the intact strand through the break
- GyrB re-ligates the break to form a negative supercoil

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18
Q

what is ciprofloxacin and its affect on DNA supercoiling?

A

it is a quinolone antibiotic that targets gyrase:
- it stabilises the covalent complex by binding to gyrase and preventing it from repairing the DNA break from GyrA
- transcription now cannot occur

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19
Q

How to topoisomerases compete in supercoiling?

A

supercoiling is a balance of topoisomerase activity:
- if there is a negative supercoil from Topo II, Topo I introduces a single-strand break and holds both ends and passes the intact strand through
- topo I then re-ligates the break to relax the negative supercoil and balance with a positive supercoil

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20
Q

what are the characteristics of bacterial transcription?

A
  • promoters consist of a -35 (TGTTGACA) and a -10 (TATAAT) sequence
  • -35 and -10 are bound by RNAP
  • in perfect promoters there are 17bps between the two sequences for RNAP binding
  • transcription starts at +1 and ends at terminator sequence
  • protein-coding transcripts include a Shine-Dalgarno sequence (AGGAGG) shortly before the ATG start codon
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21
Q

what is the structure of RNAP?

A

core polymerase contains 2 alpha, 1 beta, 1 beta’ and 1 omega subunits

22
Q

what is a holoenzyme?

A

active RNAP when sigma-factor is bound to the polymerase
- can now recognise promoters

23
Q

what is the process of transcription initiation?

A
  1. RNAP binds to DNA non-specifically
  2. RNAP scans DNA until sigma-70 recognises a promoter in the closed complex
  3. RNAP unwinds the DNA to form an open complex
  4. sigma-70 factor is removed and transcription begins
24
Q

what is the process of rho-dependent transcription termination?

A
  1. Rho protein complex recognises and binds GC-rich sequence in mRNA after ORF
  2. Rho wraps the RNA around itself - Rho ATPase activity drives spooling
  3. when rho makes contact with RNAP, transcription is terminated
25
Q

what is the process of rho-independent transcription termination?

A
  1. terminator is a stable GC-rich stem-loop in mRNA
    - stem-loop is a sequence where the G and C nucleotides of the RNA pair together and form a GC-rich loop
  2. once transcribed, contact between the stem-loop and the RNAP stops transcription
26
Q

where can gene regulation occur?

A

80-90% of gene regulation occurs at the level of transcription
- every step in this process is subject to regulation: control rate or transcription initiation, suppression of termination

translation rate can also be regulated

27
Q

how are genes regulated?

A

bacteria use regulons to control whole gene expression, or specific genes individually

28
Q

what are sigma factors?

A
  • bacteria use alternative sigma factors direct gene transcription
  • can be controlled by anti-sigma factors
  • RNAP loads with sigma factor to recognise specific promoters
  • sigma factors are only present when needed
  • some genes are controlled by more than one sigma factor and more than one promoter
29
Q

which sigma factor is dominant in a stable, healthy environment? what happens when these conditions change?

A

sigma 70 regulon is dominant in normal conditions

when conditions change, bacteria switch to use a different sigma factor to adapt to the changing conditions

30
Q

how many sigma factors does E. coli have?

A

7

31
Q

what are the sigma factors of E. coli?

A
  • σ70 – housekeeping σ-factor(σA) – responsible for transcription of majority of genes in genome
  • σ38 – general stress response σ-factor (σS) – allows bacteria to adapt to wide range of stresses e.g. lack of nutrients or oxygen
  • σ32 – heat shock/cytoplasmic stress σ-factor (σH) – adapt to changes in temperature e.g. fluidity of membranes
  • σ28 – flagellar and motility gene σ-factor (σF; FliA) – allows them to swim from threats or towards nutrients
  • σ24 – extracytoplasmic stress σ-factor (σE)
  • σ19 – ferric citrate transport σ-factor (σI) – iron scavenging
  • σ54 – nitrogen-related σ-factor (σN)
32
Q

which sigma factors have the largest regulons?

A

sigma-70 and sigma-38 have the largest regulons
- bacteria can survive with just these 2

33
Q

what is sigma-70/sigma-A?

A
  • the housekeeping sigma factor
  • their promoters have -10 and -35 elements
  • the most abundant sigma factor during exponential growth
34
Q

how is sigma-70 regulated and swapped for sigma-38 during stress?

A

2 anti-sigma-70 factors sequester sigma-70 in stationary phase of growth:

  1. Rsd binds to region 4 and blocks sigma-70 from binding to RNAP core, allowing lower affinity sigma-38 to bind to RNAP
  2. HscC acts like a DnaK chaperone to bind and sequester sigma-70 to deplete the sigma-70 pool in the cell and enable sigma-38 to take over and deal with cellular stresses
    - less specific
35
Q

what is sigma-38/sigma-S?

A
  • the general stress sigma factor
  • used when environmental stability is lost and conditions change such as temp, osmolarity, nutrients and oxygen
  • promoters have -10 and -35 sequences with 17bp spacing
  • complex regulon: switch from sigma-70 to 38 is made when absolutely necessary
36
Q

how do sigma-70 and sigma-38 differ?

A
  • sigma-38 abundance is 1/3 of sigma-70
  • sigma-38 has lower affinity for RNAP core than sigma-770
  • if 70 and 38 are present at the same time, 70 will dominate unless it has been sequestered
37
Q

how is sigma-38 regulated?

A

subject to very complex regulation:
- rpoS encodes sigma-38
- its promoter is recognised by sigma-70
- expression of this promoter is controlled by transcription factors based on the metabolic conditions of the cell
- when environment is stable, transcription factors suppress the promoter
- when environment is unstable, rpoS is transcribed and the mRNA is subject to regulation
- mRNA has a 5’-UTR region which is regulated by small RNAs to prevent its translation
- when regulation is overcome, mRNA is translated to sigma-38
- sigma-38 can be regulated by Ira proteins which direct sigma-38 for proteolysis degredation with RssB
- only when cell is extremely stressed, all regulation is overcome and sigma-38 can now bind to RNAP and control transcription

38
Q

what is sigma-32/sigma-H?

A
  • heat-shock response sigma factor
  • rpoH encodes sigma-32, which is controlled by sigma-70
  • intrinsic secondary structures in the sigma-32 mRNA are temperature sensitive, causing sigma-32 to respond to heat-shock
39
Q

how is sigma-32 regulated by temperature?

A

at low temps (<30C):
- mRNA translation is low
- translation cannot occur due to the loops in the RNA being stable, so ribosome cannot attach and less sigma-32 is expressed
- any sigma-32 that is made is sent for degradation

at high temps (42C):
- secondary structure loops in the mRNA is melted and now linear
- ribosomes can bind and translate the mRNA efficiently to produce lots of sigma-32
- sigma-32 is not degraded as the degradation proteins become denatured

40
Q

what is sigma-28/sigma-F (FliA)?

A
  • activates expression of several motility and flagellar synthesis genes
  • sigma-28 promoters have an extended -10 element (GCCGATAA)
  • sigma-28 is half as abundant as sigma-70 under most conditions, but decreases upon heat shock
41
Q

what are the components of the flagella formed by sigma-28?

A

components of the flagella must be organised in the correct order:
- motor rotates flagella inside
- hook on outside of motor
- filaments on outside of hook and can rotate

42
Q

how does sigma-28 control the flagellar assembly cascade?

A

class 2: controlled by sigma-70 regulons
- in healthy environment, sigma-70 encodes proteins which assemble the base of the flagella, the membrane motor and the hook
- in this regulon is FliA which encodes sigma-28

class 3: controlled by sigma-28
- FliC makes the filaments
- encodes motility genes to sense nutrients

43
Q

how is sigma-28 tightly controlled when forming the flagella?

A

class 2 encodes flgM which is an anti-sigma factor which sequesters sigma-28 during class 2 expression
- this ensures that the filament isn’t assembled while the hook is being made
- once the hook is made, flgM is secreted from the cell, so sigma-28 is now free to make the filament

44
Q

what are two component systems (TCS)?

A
  • TCS allow bacteria to sense and respond to their environment
  • they can controls singular genes at local level
  • they are tuned to a specific environmental condition e.g. phosphate levels
  • they consist of a sensor (histidine kinase) and an effector (response regulator)
  • some bacterial genomes contain 100s of TCS
45
Q

what is the organisation of a simple TCS?

A
  • sensor histidine kinase (HK) is membrane-bound
  • on receipt of signal, a catalytic ATPase domain binds to ATP, hydrolyses it and uses the Pi to phosphorylate a conserved histidine residue in the transmitter domain
  • therefore HK self-phosphorylates
  • the phosphorylated HK transiently interacts with its response regulator (RR)
  • the phosphoryl group is transferred to the aspartate residue on the receiver domain of the RR
  • the phosphorylated RR becomes the activated effector protein
46
Q

what is the structure of some common histidine kinases?

A
  • contains couple TMs, sensor in loop between TMs, then HK domain and catalytic ATPase domain
  • the loop between transmembrane domain is extracellular
  • can contain PAS which is a protein with a left hand groove that binds to ligands
  • GAF acts in a similar way to PAS
  • HAMP domain is a 4-helical bundle which transmits a conformational change along the protein.
47
Q

what is the structure of some common response regulators?

A
  • some RRs are just the receiver domain itself
  • most common form have a DNA-binding domain (transcription factor)
  • can be linked to AAA+ ATPase which can modulate sigma-54
  • can be linked to GGDEF for synthesis of cyclic-di-GMP which switches the bacteria from sessile to motile
  • can be linked to methyltransferases which are involved in the chemotactic reponse
48
Q

what are the roles of accessory proteins in TCS?

A
  1. can have a separate sensory protein that transmits to HK
  2. can be a scaffold protein which facilitates transmission to RR
  3. can be a connector protein which can swap signals to other nearby TCS, allowing different TCS to work together
  4. can be allosteric, so the RR feeds into another TCS
49
Q

how is the response regulator activated (RstA)?

A
  1. RstB (HK) phosphorylates monomeric RstA (RR), leading to dimerisation
  2. DNA binding by the DNA-binding domain (DBD) changes the conformation of RstA
  3. a second DBD searches for a suitable binding site in the next major groove
    - function requires 2 adjacent sites
  4. RR bound to DNA makes contact with the C-terminal domain of the RNAP alpha-subunit, or with the sigma factor, to activate transcription
50
Q

how does the regulatory circuit of acid resistance in E. coli work?

A
  • Involves TCS which senses low pH and a connector protein that feeds into σ38
  • EvgSA (TCS) senses low pH, induces expression of safA and ydeO genes
  • SafA encodes a membrane-bound connector protein that interacts with PhoQ (another TCS) – PhoQ becomes activated and phosphorylates itself and PhoP (TCS)
  • PhoP regulates one of the Ira genes (see σ38 regulation) – allows switch from 70 to 38
  • IraM sequesters RssB, preventing degradation of σ38
  • One of the σ38 regulon is gadE, transcription of which also needs YdeO
    o Acid stress allows combination of YdeO and sigma-38 to produce gadE
  • gadE -> acid response by secreting H+ ions from the cell and increasing the pH in the cytoplasm

Microbiology 2 (10 decks)

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