Prokaryotic gene regulation Flashcards
what are bacteria?
free-living organisms that need to quickly adapt to changing environmental conditions to survive:
- often live in competitive environments so must maximise efficiency
what is the typical structure of a bacterial genome?
- 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
how is the nucleoid organised in bacteria?
- 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
what are the features of the E. coli nucleoid?
- 4.6mbp genome
- a circle of DNA 1.6mm long inside a 2um cell
- approx 400 domains in E. coli
what are nucleoid-associated proteins (NAPs)?
- 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
What are the 6 NAP types in E. coli? what are their roles?
- H-NS
- Fis
- IHF
- HU
- Dps
- CbpA
what is the role of H-NS NAP?
bridges adjacent segments of DNA
what is the role of the Fis NAP?
Induces sharp bends
what is the role of the IHF NAP?
induces sharp bends
what is the role of the HU NAP?
condenses DNA into a fibre
- the most conserved NAP
what is the role of the Dps NAP?
condenses DNA to protect it from damage during stress, expressed in stationary phase
what is the role of the CbpA NAP?
condenses DNA to protect it from damage during stress, expressed in stationary phase
why is DNA supercoiling useful?
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
what are the types of DNA supercoils?
positive supercoil: over-twisting the DNA helix
negative supercoil: under-twisting the DNA helix
what controls DNA supercoiling?
topoisomerases
how many topoisomerases does E. coli have?
I-IV
- Topo I, III and IV introduce positive supercoils
- Topo II (gyrase) forms negative supercoils
how does Topo II (gyrase) form negative supercoils?
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
what is ciprofloxacin and its affect on DNA supercoiling?
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
How to topoisomerases compete in supercoiling?
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
what are the characteristics of bacterial transcription?
- 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
what is the structure of RNAP?
core polymerase contains 2 alpha, 1 beta, 1 beta’ and 1 omega subunits
what is a holoenzyme?
active RNAP when sigma-factor is bound to the polymerase
- can now recognise promoters
what is the process of transcription initiation?
- RNAP binds to DNA non-specifically
- RNAP scans DNA until sigma-70 recognises a promoter in the closed complex
- RNAP unwinds the DNA to form an open complex
- sigma-70 factor is removed and transcription begins
what is the process of rho-dependent transcription termination?
- Rho protein complex recognises and binds GC-rich sequence in mRNA after ORF
- Rho wraps the RNA around itself - Rho ATPase activity drives spooling
- when rho makes contact with RNAP, transcription is terminated
what is the process of rho-independent transcription termination?
- 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 - once transcribed, contact between the stem-loop and the RNAP stops transcription
where can gene regulation occur?
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
how are genes regulated?
bacteria use regulons to control whole gene expression, or specific genes individually
what are sigma factors?
- 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
which sigma factor is dominant in a stable, healthy environment? what happens when these conditions change?
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
how many sigma factors does E. coli have?
7
what are the sigma factors of E. coli?
- 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)
which sigma factors have the largest regulons?
sigma-70 and sigma-38 have the largest regulons
- bacteria can survive with just these 2
what is sigma-70/sigma-A?
- the housekeeping sigma factor
- their promoters have -10 and -35 elements
- the most abundant sigma factor during exponential growth
how is sigma-70 regulated and swapped for sigma-38 during stress?
2 anti-sigma-70 factors sequester sigma-70 in stationary phase of growth:
- Rsd binds to region 4 and blocks sigma-70 from binding to RNAP core, allowing lower affinity sigma-38 to bind to RNAP
- 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
what is sigma-38/sigma-S?
- 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
how do sigma-70 and sigma-38 differ?
- 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
how is sigma-38 regulated?
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
what is sigma-32/sigma-H?
- 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
how is sigma-32 regulated by temperature?
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
what is sigma-28/sigma-F (FliA)?
- 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
what are the components of the flagella formed by sigma-28?
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
how does sigma-28 control the flagellar assembly cascade?
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
how is sigma-28 tightly controlled when forming the flagella?
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
what are two component systems (TCS)?
- 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
what is the organisation of a simple TCS?
- 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
what is the structure of some common histidine kinases?
- 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.
what is the structure of some common response regulators?
- 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
what are the roles of accessory proteins in TCS?
- can have a separate sensory protein that transmits to HK
- can be a scaffold protein which facilitates transmission to RR
- can be a connector protein which can swap signals to other nearby TCS, allowing different TCS to work together
- can be allosteric, so the RR feeds into another TCS
how is the response regulator activated (RstA)?
- RstB (HK) phosphorylates monomeric RstA (RR), leading to dimerisation
- DNA binding by the DNA-binding domain (DBD) changes the conformation of RstA
- a second DBD searches for a suitable binding site in the next major groove
- function requires 2 adjacent sites - RR bound to DNA makes contact with the C-terminal domain of the RNAP alpha-subunit, or with the sigma factor, to activate transcription
how does the regulatory circuit of acid resistance in E. coli work?
- 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
