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MBIO2020 > 2.1 - Regulation of Gene Expression pt.2 > Flashcards

2.1 - Regulation of Gene Expression pt.2 Flashcards

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1
Q

what are two component regulatory systems?

A

-a system consisting of a specific sensor kinase (typically in the cytoplasmic membrane) and a response regulator protein in the cytoplasm

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2
Q

what is a kinase?

A

-an enzyme that phosphorylates compounds
-phosphate typically comes from ATP
-histidine kinase

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3
Q

what does a kinase do when it detects a signal?

A

-phosphorylates itself (autophosphorylation) at a specific histidine residue

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4
Q

what happens to the phosphate on the kinase?

A

-it is transferred from the kinase to the response regulator which activates its function as a DNA-binding protein to regulates transcription in a positive or negative manner

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5
Q

how is the response terminated to complete the regulatory circuit?

A

-response regulator gets dephosphorylated by a phosphatase
-creates a feedback loop to ensure a functional balanced regulatory system

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6
Q

how many two-component systems are present in E.coli?

A

-almost 50

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7
Q

what are examples of two component regulatory systems within E.coli?

A

-porin regulation (OmpR-EnvZ)
-pho regulon
-nitrogen utilization (Ntr) (NRI-NRII)

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8
Q

what are porins?

A

-protein channels that allow metabolites to cross the outer membrane

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9
Q

what environmental signal controls the levels of OmpF and OmpC porins?

A

-osmolarity/osmotic pressure
-low osmotic pressure (less solutes) = synthesis of OmpF increases (larger)
-high osmotic pressure (more solutes) = synthesis of OmpC increases (smaller)

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10
Q

what is the process of the OmpR-EnvZ two component system

A

-changes in osmotic pressure are detected by the EnvZ histidine kinase
-when a shift occurs, EnvZ is autophosphorylated and transfers the phosphate group to the response regulator OmpR

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11
Q

what occurs under low osmotic pressure in terms of transcription?

A

-OmpR-P activates transcription of ompF
-because you want to open up and have more water come inside the cell

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12
Q

what occurs under high osmotic pressure in terms of transcription?

A

-OmpR-P represses transcription of ompF and activates transcription of ompC
-because you want to keep water in the cell

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13
Q

what also controls expression of ompF?

A

-a regulatory RNA

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14
Q

what is the expression of ompF and ompC dependent on?

A

-the concentration of OmpR-P
-the intracellular level of OmpR-P is controlled by the dual EnvZ functions (kinase and phosphotase)
-ratios are created in response to osmolarity of the growth medium
-a lower concentration of OmpR-P will result in less binding

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15
Q

what is the process of the Ntr two component system?

A

-response regulator is NRI and the sensor kinase is NRII
-NRII regulates ammonia levels in the cell through its dual function (kinase + phosphotase) depending on the nitrogen status of the cell
-the activity of NRII is controlled by the state of signal transducing protein PII

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16
Q

what is the role of the PII protein?

A

-regulates a diverse range of transcription factors, enzymes, and membrane transport proteins

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17
Q

what controls the activity of PII proteins?

A

-if they have been covalently modified (post-translational regulation)
-uridylylation (addition of an UMP group)
-adenylation (addition of an AMP group)

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18
Q

what is ammonia assimilation?

A

-the process of turning ammonia into organic molecules (amino acids)

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19
Q

how is the necessity for ammonia assimilation determined?

A

-through glutamine sensing by GlnD (alarm in the system)
-if glutamine is low, then GlnD will transduce this signal as glutamine does not bind to the GlnD, which is what it typically does

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20
Q

how does Glnd act to cause glutamine synthesis? what is the further result of this?

A

-GlnD adds on a UMP group to PII (uridylyltransferase activity)
-PII-UMP then activates glutamine synthease by inducing the removal of the AMP group attached to it
-PII-UMP also activates kinase activity of NRII (autophosphorylation) which will then phosphorylate the response regulator NRI
-NRI-P will then activate nitrogen assimilation genes

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21
Q

why must glutamine synthease be tightly controlled?

A

-key energy requiring enzyme in ammonia assimilation
-energy must be conserved if cellular levels of nitrogen are high as sensed by GlnD

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22
Q

what happens when glutamine levels are sufficient?

A

-glutamine will bind to GlnD which activates its function to remove UMP from PII

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23
Q

what will the unmodifed PII stimulate?

A

-phosphotase activity of NRII which will result in no NRI-P and therefore no transcription

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24
Q

what is phosphorus essential for?

A

-nucleic acids (DNA + RNA)
-membrane synthesis
-energy generation
-cell signalling
-as a biological buffer to contribute to pH homeostasis

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25
how is phosphorus available?
-present as inorganic phosphate (Pi) and is often a limiting nutrient -the transformations of Pi are heavily regulated by the PhoRB two component system
26
what is the process of the PhoRB two component system?
-PhoR is a dimeric histidine kinase and phosphatase -PhoB is a response regulator that when in its phosphorylated form binds to Pho boxes within promotor regions upstream of genes -PhoR activity is controlled by PstSCAB transporter and PhoU -high environmental and cytoplasmic levels of Pi favours PhoR in its phosphotase conformation -low environmental levels of Pi favours PhoR in its autokinase conformation
27
what is PhoU?
-peripheral membrane protein that modulates Pi transport through the PstSCAB complex -acts as a brake to prevent too much Pi import
28
what is the PstSCAB transporter?
-high affinity Pi transporter (member of the ABC transporter family) -Pst Protein is a periplasmic phosphate binding protein that binds and presents phosphate to transmembrane components PstC and PstA -ATP binding across the PstB dimer interface undergoes hydrolysis to trigger a conformational change of the gate to become inward facing and allow Pi access to the cytoplasm -the cycle continues as ADP is released and ATP rebinds
29
how does the Pho regulon act in streptomyces?
-streptomyces is a gram positive bacteria that contains many species that produce antibiotics (energy intensive + limited by high phosphate conditions) -in a natural habitat it produces antibiotics to kill competing cells of other bacterial species for phosphate resources -mechanism for sensing extracellular Pi levels is unknown
30
what is quorum sensing?
-regulatory mechanism that assesses population density -widespread among gram negative bacteria, many gram positive species, and some archaea -used to ensure that sufficient cell numbers of their own species are present before initiating activities that require a certain cell density to work effectively
31
how is quorum sensing quantified?
-by a concentration of specific signal molecules produced by bacterial cells of the same species (called an autoinducer)
32
how do autoinducers act?
-typically diffuse freely across the cell envelope in either direction -high concentrations are achieved inside the cell only if many cells are nearby -function to trigger gene transcription by directly binding a transcriptional regulator or activating a two component system
33
what are the 3 different classes of autoinducers?
-those specific to bacterial species -those for interspecies communication -small peptides
34
what were the first identified specific autoinducers?
-acyl homoserine lactones (AHLs) -acyl groups are functional groups containing carbonyl and alkyl groups -AHLs of different species of gram negative bacteria feature different acyl groups of different lengths
35
what is an autoinducer used for interspecies communication among gram negative bacteria?
-cyclic furan derivative AI-2
36
what species use small peptides as autoinducers?
-gram positive bacteria (staphyl) -some archaea
37
what is an example of an autoinducing peptide in staphylcoccus aureus?
-AIP -basal transcription of ArgD is pre-AIP -processing of ArgD into AIP by ArgB (to differentiate intracellular and extracellular versions) -AIP triggers the two component system of ArgC/ArgA which activates the expression of virulence genes
38
how does the shiga-toxin producing E.coli use autoinducers?
-has 3 molecules that activate virulence gene expression via two component systems -levels of AI-3 increase with more E.coli -stress hormones epinephrine and norepinephrine from host intestinal cells -the virulence genes lead to toxin production, bacterial motility, and lesions of intestinal cells to release nutrients
39
what is Aliivibrio fischerii?
-bacterial symbiont of marine animals including squids (inhabit the squids light organ to mimic moonlight to hide from predators) -proteins needed for their luminescence are encoded by the lux operon under the control of activator protein LuxR -gets induced by a threshold concentration of specific A.fischerii AHL
40
what is chemotaxis?
-behaviour of cells moving towards attractants and away from repellents -cells sense a change in concentration of a chemical over time (not absolute concentration) -sensed information regulates the direction of flagellar rotation -ex: pseudomonas aeruginosa cells moving towards epithelial cells to scavenge nutrients (damaging them in the end)
41
what does the mechanism of chemotaxis depend on?
-a signal cascade of multiple proteins -sensory proteins called methyl accepting chemotaxis proteins (MCPs) sense attractants and repellents and interact with cytoplasmic sensor kinases
42
what do the MCPs form?
-large hexagonal arrays known as chemoreceptors
43
where are chemoreceptors located?
-in the cytoplasmic membrane and/or cytoplasm -ex: vibrio sp. has transmembrane and cytoplasmic chemoreceptors -ex: E.coli has onlu transmembrane chemoreceptors
44
what chemoreceptors does E.coli have?
-4 transmembrane ones -each is composed of 5 different MCPs that are specific for certain compounds -ex: Tar MCP senses the attractants aspartate and maltose and the repellents cobalt and nickel
45
how do MCPs bind attractants/repellents?
-directly
46
what cytoplasmic proteins get triggered when an attractant/repellent binds to an MCP?
-CheA (sensor kinase) -CheY (response regulator)
47
what response is initiated when MCPs bind an attractant or release a repellent?
-a coupling protein called CheW is inactive and autophopshorylation of CheA is inhibited -cell moves in a run and swims smoothly because there is no binding of CheY (counterclockwise run)
48
what response is initiated when MCPs bind a repellent or release an attractant?
-conformational change occurs and CheA is autophosphorylated -CheA-P phosphorylates CheY -this causes a clockwise flagellar rotation (tumbling cells) -CheZ dephosphorylates CheY to return cells to runs
49
what must occur after the stimulus of a repellent or attractant has been responded to?
-the sensory system needs to reset to await for further signals (adaptation)
50
what in MCPs allows for adaptation to sensory signals?
-varying methylation -fully methylated MCPs can no longer respond to attracts (still sensitive to repellents) -unmethylated MCPs respond strongly to attractants (insensitive to repellents)
51
what controls the methylation of MCPs?
-chemotaxis protein CheR methylates MCPs -chemotaxix protein CheB-P demethylates MCPs
52
what occurs in the presence of a high level of attractant?
-the rate of autophosphorylated CheA is low, leading to unphosphorylated CheY and CheB -cell is in a smooth run -the methylation of MCPs increases during the second period because CheB-P is not present to demetthylate (since no Che-A-P) -fully methylated MCPs no longer response to an attractant (at a constant level) so the attractant is released and CheW helps CheA-P> CheyY-P and CheB
53
what occurs in the presence of a high level of repellent?
-the rate of autophosphorylated CheA is high, causeing the CheY-P to tumble the cells and CheB-P to demethylthate -fully methylated MCPs respond best to an increasing gradient of repellents (send a signal for cell tumbling while MCPs are slowly demethylated
54
what does chemotaxis achieve?
-the ability to monitor small changes in concentrations (gradients) of both attractants and repellents over time
55
what are non-coding RNAs (ncRNA)?
-RNA molecules that do not get translated to polypeptides -have no shine dalgarno sequence -ex: tRNA, rRNA, small RNAs (sRNAs)
56
what is the role of sRNAs?
-regulate gene expression -base pair directly to other RNA molecules (mRNAs) that have complementary regions -modulate the translation rate of the target mRNA -ex: in E.coli a number of sRNA molecules regulate various cell physiology aspects in response to environmental or cellular signals by binding to other RNAs or other small molecules to control gene expression
57
what are the 2 mechanisms that sRNAs do to affect changes in protein expression for the target mRNA?
-change its secondary structure to block a previously accessible ribosome binding site (bind to complementary base pairing in the RBS) = decrease protein expression -open up a previously blocked ribosome binding site to allow access for the ribosome = increase protein expression
58
what are the 2 mechanisms that sRNAs do to affect mRNA stability to increase or decrease degradation by ribonucleases and thus modulate protein expression?
-sRNA interaction decreases mRNA stability = less protein expression + increase degradation -sRNA interaction increases mRNA stability = more protein expression + decrease degradation
59
what is the MicF sRNA?
-one of the first sRNAs discovered -part of it is complementary to the 5' end of ompF gene including the translation initiation region -the expression of micF is under control of four transcriptional activators including OmpR
60
what is a riboswitch?
-RNA molecules that specifically recognize and bind other molecules including low molecular weight metabolites -affect gene expression through transcriptional or translational control depending on the type of switch
61
what does the binding of metabolites to riboswitches require?
-folding of RNA into a specific 3D structure that recognizes the target molecule (pocket) -does not require complementary base pairing
62
what are riboswitches commonly found to control?
-synthesis of enzymes in biosynthetic pathways for various vitamins, few amino acids, some nitrogen bases, and for a precursor in peptidoglycan synthesis
63
what is an example of a riboswitch in the thiamine operon?
-vitamin B1 -3 regions (3rd = shine dalgarno sequence) -if the levels of thiamine are low in the cytoplasm then regions 1 and 2 will base pair and the shine dalgarno sequence will be accessible for translation -if levels of thiamine are high in the cytoplasm, then the metabolite (thiamine pyrophosphate) binds to region 1 to prevent the 1:2 hairpin -a second hairpin forms (2+3) to make the shine dalgarno sequence inaccessible for translation + mRNA is eventually degraded
64
what is an example of a riboswitch in SAM in Bacillus subtilis?
-cofactor in methionine biosynthesis pathway -when SAM levels are low the leader sequence forms the correct hairpin and transcription continues as normal -when SAM levels are high the SAM binds to the hairpin structure and induces formation of the termination structure (hairpin followed by a poly U tail) (premature intrinsic terminator)
65
what is the stringent response mechanism?
-widely distributed regulatory mechanism used by bacteria to survive nutrient deprivation and environmental stresses -balances the metabolic state of the cell -activation of the mechanism results in shutdown of macromolecule synthesis and the activation of survival pathways -responsive to decreased availability of amino acids (moving from a rich complex medium to a defined medium with a single carbon source)
66
what happens when the growth of the cell is limited by a shortage of amino acids?
-the pool of uncharged tRNAs increases relative to the charged tRNAs -uncharged tRNAs get inserted into the ribosome and results in stalling which leads to (p)ppGpp synthesis by RelA (alarm)
67
what triggers the stringent response?
-an accumulation of unusual nucleotide guanosine tetraphosphate (ppGpp) (alarmones) -accumulate during stress or during a shift down from amino acid excess to amino acid starvation
68
how is ppGpp made?
-first made as guanosine pentaphosphate (pppGpp by transferring two phosphates from ATP to the 3' OH of GTP (done by RelA) -quickly converted to ppGpp by phosphatase Gpp
69
how does RelA act?
-RelA is associated with the 50S ribosomal subunit and gets activated by a signal from the ribosome during amino acid limitation -ex: translation of one codon occurs normally, translation of the next codon does not have a aminoacylated tRNA available -peptidyl-tRNA in the P site and an empty A site -if the ribosome is stalled long enough then the unaminoacylated tRNA binds to the A site (without EF-Tu-GTP) (activates RelA)
70
what controls intracellular levels of ppGpp along with RelA?
-SpoT -bifunctional (has pppGpp synthesis activity + hydrolase activity to degrade ppGpp) -does not associate with ribosomes (monitors changes in carbon metabolism through direct interaction with acyl carrier protein (ACP))
71
what is the role of ACP?
-fatty acid biosynthesis by binding to short-chain fatty acids and accepting additional fatty acid substrates to build longer fatty acid chains
72
what are the affinities to SpoT when ACP is bound by different fatty acid chain lengths?
-ACP bound by long-chain fatty acids have low affinity to binding SpoT -ACP bound by short-chain fatty acids has high affinity to binding SpoT which stimulates its dual functions
73
when is carbon metabolism not functioning efficiently?
-when there is shorter chain fatty acids
74
what changes occur in the cell from the stringent response?
-synthesis of rRNA and tRNA ceases almost immediately -protein and DNA synthesis is reduced, but biosynthesis of new amino acids is activated (synthesized by proteins made by existing ribosomes) -eventually synthesis of rRNAs and in turn ribosomes resumes but at a new rate to reflect the cell's reduced growth rate
75
what is an example of how the stringent response occurs in E.coli cells?
-switching environments from nutrient rich intestine to an open water system -the reduction in nutrients triggers synthesis of ppGpp
76
what is an example of how the stringent response occurs in C. crescentus cells?
-naturally inhabit nutrient-poor environments -not an amino acid limitation, but instead a carbon and ammonia starvation -this triggers a switch from stalked to swarmer cells (higher levels of ppGpp)
77
what is an example of how the stringent response occurs in the infected human lung?
-lung is low in oxygen and limited for phosphate along with exposure to host immune system induces ppGpp production -this promotes the formation of relatively dormant persister cells that have antibiotic resistance and granulomas -once aerosolized and relieved of stress the cells revert back to infective vegetative cells
78
what is the general stress response?
-a stationary phase of growth -controlled by activating the RpoS regulon which has over 400 genes -recognized by the alternative sigma factor RpoS (sigma S or 38 or stationary sigma factor) -has transcriptional, translational, and post-translational regulatory mechanisms
79
80
what regulates the translation of rpoS transcripts?
-positively regulated by sRNAs that are expressed during stress conditions -RpoS is susceptible to degradation during non-stressful conditions
81
what types of genes are on the RpoS regulon?
-nutrient limitation -resistance to DNA damage -biofilm formation -responses to osmotic, oxidative, and acid stresses
82
what factors can cause protein denaturation?
-temperature -ethanol -UV light -osmotic stress
83
what is the role of heat shock proteins?
-help counteract protein damage and assist in the cell in recovering from stress -grouped into a regulon controlled by alternative sigma factor RpoH (32)
84
what degrades RpoH under normal conditions? what prevents degradation under heat shock conditions?
-DnaK -in heat shock conditions DnaK is busy with folding denatured proteins leaving RpoH alone to complex with RNA polymerase for transcription initiation of heat shock genes

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