Topic 1 - Bacterial gene regulation Flashcards
Features of organisation of the E. coli chromosome.
Singular circular chromosome
Few introns with no spliceosome (self-splicing)
DNA is mostly coding
supercoiled
why do bacteria need such quick changes in gene expression in contrast to somatic cells
they are free living so must adapt to environmental changes evolution has selected for efficiency and flexibility above all else
Why do transcriptional units tend to be orientated in the same direction as chromosome replication
avoids crashes between polymerases
which NAP bridges DNA
H-NS
which NAPs bend DNA
Fis and IHF
which NAP condenses DNA
HU
what does the GyrB/GryA complex do
GryB binds DNA
GryA is a ATPase which hydrolyses DNA introducing a DOUBLE strand break and passing the intact strand through the break
GryB religates the break
this results in a negative supercoil
this is a target for quinolone antibiotics
which enzyme relaxes negative supercoils? how does it work?
topoisomerase I (Topo I)
SINGLE strand break passes intact strand through to decrease the number of coils and relax the supercoil
what subunits make up a RNA polymerase
2 alpha
1 beta
1 beta’
1 omega
+ a sigma factor to make it a holoenzyme
describe the upstream promoter
-35 site = TGTTGACA
-10 site = TATAAT
In perfect promoters there are 17bp between the two
So less stable promoters can be made by changing this
what is the Shine-Dalgarno (SD) sequence (seq and purpose)
AGGAGG - binds to ribosomal RNA before the initiating ATG codon.
what is the start codon
ATG
what is Rho dependant transcription termination
Rho binds to an exposed region of RNA after the ORF segment at GC-rich sequences that lack obvious secondary structure
It assembles a hexamer and wraps around mRNA faster than the new strand is being produced until it hits the polymerase freeing it
What is Rho independent transcription termination?
Based on the mRNA structure; requires a palindromic GC-rich region, forms a stem loop structure, contracts RNA polymerase and stops transcription.
what relaxes and forms supercoils
topoisomerases
topology - shape
isomerise change
how many topoisomerases does E.coli have
4
which is the only topoisomerase in E.coli which can induce supercoils (negative)
2
what is the default way of supercoiling bacteria have chosen
negative
what is σ70
housekeeping functions
what is σ38
stress response
what is σ32
heat stress response
what is σ28
flagella
what is the σ28 σ factor cascade
complex machinery, important to build it in the right order
class 1, class 2 by σ70, and 3 by σ28
genes expressed sequentially in the cell
then proteins assembled in the right order
basal body –> hook –> filament
antiσ28 - FlgM in class one, kicked out in class 2
what is σ54
nitrogen
what do two component systems do
sense and respond to an environmental change
what is the sensor of a two component system
a histidine kinase
what is the effector of a two component system
the response regulator
what are the components of a histidine kinase
On receipt of the signal the CA (catalytic ATPase) domain binds ATP and phosphorylates a conserved histidine residue in the transmitter domain (DHp = Dimerisation with Histidine that is phosphorylated)
what are the components of the response regulator
The receiver domain (RD) get the phosphoryl group transferred onto it from the DHp of the histidine kinase
what are some examples of things which can be bound to to the response regulators
most commonly - DNA binding domain
AAA+ ATPase to modulate the sigma54 system
GGDEF for synthesis of c-di-GMP (sessile-motile switch)
Methyltransferases often involved in chemotactic response
Some RRs are just the receiver domain - function unclear
what are 4 types of accessory proteins to two component systems
- a separate sensor protein which transmits to the histidine kinase
- a scaffold protein which facilitates transmission from the hk to response regulator
- a connector protein between different TCSs for them to work together
- allostery - an RR without an output (just feeds into another TCS)
how have multiple TCSs evolved
gene duplication leads to to multiple TCSs which specialise and gain fidelity
Acid responce TCS
accessory sensor protein senses low pH and activates HK the regulator regulates a gene preventing sigma 38 degradation lead to the transcription of acid response genes
how can HK-RR phosphotransfer specificity be switched
mutating specific determining residues
what is a riboswitch
a regulatory sequence in mRNA which can bind a small molecule to change its expression; this doesn’t require a regulatory protein.
Usually binding decreases expression
where are riboswitches primarily found
in the 5’ UTR of mRNAs
in what organisms are riboswitches found?
all forms of life but most commonly gram +ve bacteria
what domain of riboswitches binds to the specific ligand
Aptamer domain
what is the part of sequence of mutually exclusive conformations allowing expression or not in a riboswitch
the expression platform
what is the sequence which switches domain when a ligand binds to the riboswitch
the switching sequence
when no ligand it makes up the expression platform
when ligand binds it switches to forming the aptamer domain
what are the 3 mechanisms by which riboswitches control prokaryotic gene expression
Transcriptional attenuation (premature termination of transcription)
Occlusion of the Shine-Dalgarno sequence (prevents translation)
Catalytic activity (Ribozymes)
what are the 4 types of ribosome attenuator
metabolite binding
tRNA binding
ribosome stalling
terminator/anti-terminator proteins
what is the most conserved nucleoid associated protein, it condenses DNA into a fibre
HU
how is the RNA polymerase holoenzyme different to a “normal” RNA polymerase
a RNA polymerase holoenzyme includes the sigma factor - making it the complete enzyme
how does RNA form 3D structures
intramolecular base pairs like DNA
but can include non-canonical base pairs as RNA is less rigid (permits the formation of alternative secondary structures) in this
pairs can be far apart in primary sequence
consequences for gene regulation eg. accessibility to translated which adds function
also tRNAs
what are the 2 different types of transcription termination
rho - dependent
a protein (rho) is transcribed which wraps around mRNA and knocks off the ribosome
rho - independent
no protein involved, a stable GC rich stem loop is transcribed and contacts the ribosome, stopping transcription
how can a riboswitch regulate expression
binding of small molecules modulates expression
eg. a metabolic binds when in abundance to reduce expression of a protein(s) in its pathway
what is a riboswitch
regulatory sequence in 5’ UTR - can bind small molecules
components of the riboswitch
aptamer domain - binds to small molecule changes switching sequence conformation
switching sequence - part of the aptamer domain or expression platform depending on on/off conformation
expression platform - has mutually exclusive conformations - expression or not
what is an example of a metabolite binding riboswitch?
what mechanism is this?
SAM-I upstream of gene involved in the biosynthesis of methionine and cysteine specifically s-adenosyl-methionine (precursor)
binds s-adenosyl-methionine
attenuation
what do T-box riboswitches do? what do they bind?
what mechanism is this?
sense the amount of unloaded tRNA - this is a result the corresponding AA level
lots of unloaded = more AA needs to be made - express biosynthesis genes
attenuation
mechanisms by which riboswitches can affect transcription
- (premature) attenuation
- occlusion of the Shine Dalgarno sequence
- ribozymes
thiamine pyrophosphate riboswitch, what mechanism how does it work?
occlusion of the Shine Dalgarno sequence
binding of TPP means the Shine Dalgarno sequence is no longer accessible
TPP is a cofactor for enzymes in carbon metabolism
what is antisense RNAs
a small RNA (sRNA) molecule which are complimentary to specific “sense” mRNAs
how do RRs of TCS activate gene expression
bind DNA and interact with alpha-subunit of RNA pol
OR
bind DNA and interact with sigma factor
how do RRs of TCS repress gene expression
bind DNA in middle of promoter
blocks RNA pol from binding
what are the 6 ways antisense small RNAs can alter bacterial gene expression
transcription interference/attenuation
substrate for endo/exonuclease altered
translation blocked directly or indirectly
what strategies can bacteria use for phase variation
slipped strand mispairing
methylation
invertible gene switches
why would bacteria what to use phase variation
as a bet hedging strategy - they don’t know what environment they will face next if some make a protein, some not, at least some of a population will survive
what are the effects of slip strand mispairing depending on where it occurs
regulatory region - some subtle changes in expression more likely
promoter region - alters promoter activity
change with gene - disrupts the reading frame
What are operons?
Regulatory gene series which are expressed in groups/ simultaneously.
What is the term used for the mRNA produced by operons of multiple genes?
Polycistronic mRNA (This is later spliced into separate mRNA transcripts which are individually translated)
What is the nucleoid?
The irregularly shaped region within prokaryotes containing the endogenous prokaryotic genome.
What are NAPs?
Nucleoid associated proteins, that bind to the DNA to form domains of increased packing
What NAPs condense DNA to protect it from damage during stationary phase?
Dps and Cbp
Approximately how many base pairs per turn are there in unsupercoiled (relaxed) DNA?
10bp per turn
What is the impact of Quinolone antibiotics on the GyrB/GyrA complex?
They stabilise the covalent complex (once cleavage ahs occured), preventing the religation of the DNA, indirectly introducing double stranded DNA -> killing the bacteria.
What is the role of Topoisomerases in gene switching?
Transient Topoisomerase relaxation of supercoils facilitates binding of transcription factors to the DNA and/or RNA polymerases -> facilitating transcription.
What is Rho?
Rho is an essential transcription factor protein that stops transcription
Why are all steps of transcription and translation regulated?
To ensure efficiency and agility to survive adversity in competitive environments (wasted gene expression = wasted energy)
What is the common similarity between the 3 riboswitch mechanisms?
In all of them, the binding of a small metabolite induces change to the secondary structure of the mRNA.
Summarise the general Transcriptional Attenuation Mechanism:
The metabolite is plentiful -> this binds to the mRNA as it is being transcribed -> a terminator loop forms, leading to the premature termination of translation -> the regulated gene is not expressed.
Summarise the general Occlusion of the Shine-Dalgarno Sequence Mechanism:
The metabolite is plentiful -> binds to aptamer -> this blocks the SD sequence -> inhibits translation -> no expression
What is meant by the specifer loop?
The region of the mRNA stem which binds with the tRNA anti-codon.
What is GlmS?
GlmS is an enzyme involved in the synthesis of glucosamine-6-phosphate (GlcN6P) -> when GlcN6P concentrations are high the metabolite will bind to the mRNA, inducing a conformational change in which the the mRNA self-cleaves, no longer capable of translation -> decreasing GlmS levels.
What factors are at the top level of gene expression?
Sigma Factors.
What are sigma factors?
Sigma factors are proteins that bind to RNA polymerase’s core enzyme and make it specific to a promoter -> giving the RNA polymerase its specificity .
How many sigma factors does E.coli have?
7
What are E.coli’s primary sigma factors?
Sigma Factor 70 (sigma A, controlling genes necessary for growth and normal function) and 38 (sigma S, regulating general stress response occurrence in stationary phase. )
What is flawed about the lab based process of closed batch culture?
The environment of closed batch culture, generating results for Growth/lag/deaths phases, is purely lab based and wouldn’t naturally occur.
Which E.coli sigma factors are mutually exclusive?
Sigma 70 and Sigma 38, whereas some others operate in parallel.
What are 3 methods used in response to sigma 32 to respond to heat stress.
Synthesis of Heat-shock proteins, Chaperones and increasing the degradation of denatured proteins
What is is the role of histidine kindase?
Histidine Kinase (HK) is a transmembrane signal transductor, responding to ligand binding.
What is an example of a regulatory circuit?
Acid resistance circuit in E.coli (The phoQ and SafA systems)
What is the acid resistance circuit in E.coli?
GadE is expressed to negate the negative effects of decreased pH.
EugSA senses decreased pH -> expresses safA and ydeO -> SafA converts PhoQ into PhoP which then expresses the gene iraM -> IraM sequesters RssB -> decreasing degradation of RpoS -> activating transcription factor (YdeO) -> activate gadE.
How do two component systems evolved?
There’s a single ancestral TCS -> gene duplication occurs leading to two copies of the pathway (redundant) -? over time the accumulation of mutations results in specificity-determining residues, insulating the two pathways,
What are sRNA molecules?
Small RNA molecules of 10-30 bps used in the regulation of translation
Outline Transcription Interference by sRNA with example(s):
A gene’s (MccA in C. acetobutylicum controlling conversion of methionine into cysteine) promoter is controlled by a riboswitch, there will be promoters present in convergent directions either side of the operon, during certain conditions these will both be simultaneously bound to polymerases, causing them to crash upon convergence of the transcription forks, leading to both falling off.
RNA Thermometers:
The heat resistance of RNA molecules is determined by their intrinsic structure, therefore changes in structure can occur (activating/inhibiting) dependent on changes to temperature or local chemical environment.
What’s an example of RNA thermometer:
Sigma factor 32 -> at high temperatures the stem-loop degrades, unblocking the SD sequence and allowing the expression of heat-response proteins.
What is phase variation:
A form of antigenic variation that allows for the survival of a species by expressing multiple behaviours/strategies simultaneously by the random expression particular genes within a total population.
What is slip strand mispairing?
A form of phase variation; A repetitive DNA sequence “slips” during replication, resulting in the increase/decrease of “AT” units following excision repair.
Why isn’t slip-strand mispairing epigenetic?
Slipstrand mispairing can be inherited (e.g. in huntingtons disease)
What is an example of phase variance induced by slip-strand mispairing?
OipA gene in Heliobacter pylori -> the OipA gene when expressed subverts the host’s immune response leading to the formation of ulcers and cancer, when slippage occurs in the coding region, a frame shift occurs preventing the bacteria’s pathogenicity.
What is the function of methylation in phase variation?
Methylases methylate the DNA, this has multiple functions:
- Strand recognition -> specific methylation patterns differentiate the DNA from foreign DNA
- Differentiate between the Template DNA and the newly synthesised daughter DNA during excision repair.
-Methylation affects the binding of NAPs as some can only bind to methylated DNA and vice versa.
What is an example of phase variance induced by methylation?
E.coli has 2 methylases (Dam and Dcm), DNA adenine methyltransferase randomly blocks binding of NAPs to GATC motifs, blocking binding of transcription factors and expression
-> Specifically the of the virulence factor P fimbriae ~ expression of the P Fimbriae causes the tight attachment to epithelial cells -> LRP sites for gene are 4-6 and 1-3 are exclusive -> hemimethylation causes LRP binding to site 5, liberating sites 1-3 -> liberating the pap promoter for expression.
What is LRP?
Leucine Responsive Protein binds to sites on the DNA to regulate promoter regions, inhibiting expression when the cell is unmethylated or hemi methylated LRP binding site 5
What are invertible gene switches in phase variation?
Invertible pieces of DNA bound by inverted repeat regions which allow for gene expression in one orientation, but not in the other,
How are inverted gene switches flipped?
The inverted repeats are aligned in parallel upon DNA bending, recombination then occurs, reversing the orientation of the sequence.
What are examples of inverted gene switches (and their key difference)?
Type 1 fimbriae (fim B, E, A, and the switch Region fim S) in E.coli and Cwp V in C. difficile; fim mechanism is basically fim B and E inactive and fim A is active when gene expressed, opposite is true when not, because the fim S contains the promoter region. In the Cwp V mechanism the promoter is not in the invertible gene switch, instead being upstream, with the invertible region forming a premature termination loop during the off conformation.
What enzymes catalysed the flipping of orientation of invertible gene switches?
Recombinases (specifically RecV / recombinase 5 in C. difficile)
