Regulation Flashcards
Modes of regulation
- transcriptional
- translational
- post-translational
Transcriptional
- alternative sigma factors
- postive or negative control
Translational
- regulatory RNAs
- Alt RNA 2 prime structures
Post-translational
- Protein-protein interactions
- Protein degradation
- Feedback inhibition
- Covalent modifications
Operon
Group of genes that are transcribed as a single mRNA and controlled by one promoter
Genes in the same _________ are usually involved in the same pathway
operon
Regulon
More than one gene or operons under the control of a single regulatory protein (sigma or transcription factor)
Ex: So0A Regulon system, or your emergency response system that turns on all the associated operons needed
The more complex the habitat of a bacterium, the more _________ ________ it has
sigma factors
Anti sigma factor
a protein that binds the sigma factor and prevents it from interacting with the RNA polymerase
How do sigma factors get regulated?
- transcription levels of the gene encoding the sigma factor
- post-translational modifications
- interaction of the sigma factor with its anti sigma factor
Housekeeping genes
a baseline set of genes that are required for normal cell metabolism, growth, replication, division and maintenance
Sigma factor
a protein subunit of bacterial RNA polymerase that plays a crucial role in initiating transcription by specifically binding to promoter regions on DNA, essentially directing the RNA polymerase to the correct location on the DNA to start transcribing a gene
Regulatory proteins bind DNA in a ________________ dependent manner
sequence
Helix-turn-helix motif
one common structure used to ID and bind a specific DNA sequence
What alters gene expression by binding to DNA?
Regulatory proteins
INTRACELLUAR AND EXTRACELLULAR SIGNALS CANNOT BIND DNA DIRECTLY
Recognition helix
identifies a specific DNA sequence
No ________ bonds are formed between protein and DNA
covalent
Transcription factors often bind as a _______________ or a teramer
dimer
Negative control
binding of a regulatory protein (repressor) upstream of a gene blocks transcription
Positive Control
binding of a regulatory protein (activator) upstream of a gene enhances transcription
Repressor
a protein that binds an operator and blocks transcription
Activator
a protein that binds to an activator sequence and increases
transcription
Co-repressor
molecule that binds a repressor and makes it stick to the
operator
Inducer
molecule that induces transcription
– Inducers induce by:
removing a repressor or
activating an activator
Riboswitches and small RNAs can regulate _____________ of a specific mRNA or alter its ________ ______
translation
half life
Riboswitches
Part of mRNA (5’UTR), they bind small metabolites and alter expression of the attached mRNA as a consequence
Riboswitch bonding a small molecule uncovers RNS allowing translation to occur
Small regulatory RNA’s
Often antisense (complementary to mRNA) and binding to mRNA may alter translation
Protein modification
covalently linked small molecule, such as physphoryl group - leads to conformational change in the protein which changes its activity
phosphorylation
Pro-proteins
protein is inactive until the “pro” sequence is cleaved
Phosphorylation
addition (covalent linking) of phosphate group to Ser, Thr, His and other amino acids
Post translational modifications
- phosphorylation
- redox state change
- acetylation
-methylation
-glycosylation
Phosphorylation of a transcription factor
modifications can lead to conformational changes in protein structure
Response regulator PhoB
activated in E.coli when inorganic phosphate in the environment becomes scarce - recognizes and binds a specific DNA sequence
Degradation
Stressed cell
- membrane stress activates RseP and DegS protease to release RseA/sigE from the membrane
- RseA is then further degraded by ClpXP protease
- SigE is then free to bind RNAP
In the E.coli SigE regulation example RseA is an example of a:
a. respinse regulator
b. Inducer
c. repressor
d. anti-sigma factor
e. transcription factor
d. anti sigma factor
Cell regulates its metabolism by regulating ________ __________, mRNA translation and protein function (translational
gene expression
Small RNAs and riboswitches can regulate post-translationally by hiding or reveling the _____ or __________ ____________ __________
RBS
altering transcript stability
Two component regulatory systems
- sensor kinase
- response regulator
Sensor kinase
often an integral membrane protein with the ability to transfer a phosphoryl group on to another protein (kinase activity - adds P)
Response regulator
a DNA binding protein in the cytoplasm, activated by transfer of a phosphoryl group from its sensor kinase
Homologs
two genes that are similar in sequence and share a common history (share a gene ancestor)
Orthologs
Two genes found in two different organisms that share sequence similarity and perform the same function
Paralogs
Two genes (within an organism) that arose from gene duplication and subsequently diverged or HAVE DIFFERENT sequence and function
How do Two component regulatory systems work?
- sensor kinase detects a specific external environmental signal, such as changes in pH, nutrient level osmotic pressure, cell-to-cell signals, antibiotics
- This activates the response regulator to transcribe genes to help deal with the specific stress or threat
- Response regulator may also control gene clusters that contribute to cell growth, virulence, coordinate population level responses, development systems
2 important sigma factors for E.coli
- TTGACA (major sigma factor for normal growth)
- TNTCNCCTTGAA (heat shock response)
Heat shock protein (HSP) show transiently ____________ expression as a result of a temp up shift
enhanced
Classes of heat shock proteins
- molecular chaperones
- ATP dependent proteases
molecular chaperones
- GroEL, Hsp60, GroES, DnaK
Bind and aid in refolding or recycling misfolded proteins
ATP dependent proteases
- Lon FtsH, Clp
destroy (recycle) damaged proteins
Regulation of sigma 32
- activity of sigma 32 is regulated at the level of 1. transcription 2. translation 3. post-translation
rpoH gene has ___ promoters: 3 are recognized by sigma 70 and sigma ____
4
32
The number of sigma 32 at 30C is _____
low (<50 molecules)
Heat shock _______ rpoH mRNA secondary structure - allows ribosome binding and ___________
translation (thermosensor)
During heat shock, sigma 32 levels increase _____ to ____ fold bus only for a short period of time
15 to 20
Inherently unstable protein
Sigma 32 degradation is mediated by chaperones DnaK and DnaJ (members of the heat shock regulon)
rpoH mRNA
increase in temp melts secondary structure of 30S ribosome subunit -> pre-initation complex formation
SOS genes
- mostly DNA repair
Intracellular signals
- alarms
- second messengers
Alarms
small molecules that are synthesized when a cell is stressed
- cAMP, cyclic-diGMP, ppGpp
Negative Control
- repressor is bound -> transcription is blocked
- repressor unbinds with the help of inducer (allolactose) -> transcription proceeds by the RNA polymerase
E. coli prefers using _______ over other sugars
glucose
Catabolite repression allows a global regulation to _________ the expression and synthesis of __________ to metabolize other _________ (maltose, lactose) even if they are available
suppress
proteins
sugars
cAMP (second messenger) synthesis is inhibited by _______
glucose
- glucose suppresses the activity of adenylate cyclase, the enzyme responsible for producing cAMP
When glucose is used up ……
- cAMP levels rise
- cyclic AMP receptor protein binds cAMP, in this activated form CRP can bind specific DNA sequences near promoters upstream of various operons
- > 300 genes regulated by CRP
- CRP protein -> bind cAMP -> CRP and cAMP recruits RNA polymerase for transcription
Ribosome stalling (Nutrient deprivation)
uncharged tRNA enters the A site, activating ribosome protein L11 or ribsome-associated relA.
Both proteins synthesize ppGpp (transferring pp from ATP to GTP)
ppGpp then binds RNA polymerase - immediately shuts down transcription of ribosomal components but UPREGULATES some metabolic genes (particularly genes for amino acid synthesis)
Stringent response
L11/RelA complex -> makes ppGpp ->ppGpp binds RNAP -> this stops Dna replication, stable RNA, ribosomal proteins, fatty acids BUT enhances sigma S -> sigma s enhances glycolysis, stasis survival genes, oxidative stress genes, osmotic stress genes
Biolfilm formation
- cyclic-di-GMP
- intracellular messenger found in bacteria
- regulates biofilm formation, motility, virulence, the cell cycle, differentiation and other processes
- synthesized or broken down by GGDEF and EAL domain proteins, respectively
- c-di-GMP binds transcription factors and riboswitches to modulate gene expression
What is biofilm?
Assemblage of microorganisms and their associated extracellular products at an interface or attached to a surface
- “Slime” (also flocs, aggregates)
- most microbes grow attached to surfaces
- microbes display social behavior and communicate
Examples of signals
- heat
-DNA damage - nutrient availability
Examples of second messengers
- ppGpp
-cAMP - c-diGMP
Second messengers to gene ->
- transcription factor
- alternative sigma factor
Second messengers to mRNA ->
- riboswitch
-small RNA
-inherent stability
Second messengers to protein ->
- modification (phosphorylation, methylation, etc.)
- interaction (with other proteins)
-processing
Bacteria can sense ______________ signals (chemical changes) through __________ in the cell membrane, they can also sense environmental changes by how those changes effect intracellular molecules
environmental
proteins
Two component systems are diverse and are used to sense an environmental signal then transmit that signal to a ____________ ___________(response regular) in the cytoplasm to alter gene expression
transcription factor
What are the levels of the heat shock sigma factor regulation?
- transcriptional
- post transcriptional
- post translational
Catabolite repression, mediated by _______, allows E. coli to first use its preferred carbon and energy source (_________) before producing catabolic proteins to use other sugars (_________,_______)
cAMP
glucose
lactose, maltose
ppGpp is produced in response to __________ ________ and binds directly to RNA polymerase to slow ___________
nutrient stress
metabolism
c-di-GMP can bind transcription factors and regulates diverse responses such as _________ formation
biofilm
Bioluminescence (1950)
generation of light by living organisms
- Ex: mycena, noctiluca, bacteria, dinoflagellates, fungi, shrimp, fireflies, glow worms etc
- different systems evolved independently but all require O2
Light organs
- very high population densities of bacteria in light organs
- Euprymna scolopes, Hawaiian bobtail squid
- only emit light when the bacterial population is very dense, such as inside a light organ
Bioluminescent proteobacteria - The biochemical reaction and enzymes involved were quickly discovered and described …….
during this reaction, the electrons in the flavin ring are excited and move to a higher orbital
light is emitted when they fall back to ground state
Aliivibrio fischeri
a bioluminescent, gram negative, gamma-proteobacterium originally isolated from ocean waters
- stops producing light when introduced into a fresh medium
- eventually it begins producing light again (autoinduction)
How to regain light from Aliivibrio fischeri?
- take conditioned medium from a light-producing culture
remove all cells, and add the medium to a culture of A. fischeri that is not producing light, suddenly the “dark culture” produces light - IC biochemist characterized the light-inducing chemical called “autoinducer” acyl HomoSerineLactone
Autoinducer (HSL) of A. fischeri
- synthesized by Luxl (in the lux operon)
- It freely diffuses across the cell membrane
At low cell density, it diffuses away
At high cell density, it accumulates, concentration in the cytoplasm increases and binds the LuxR transcription activator leading to increased expression of the lux operon and increased expression of LuxR
LuxR
transcription activator, when bound to its inducer HSL, LuxR binds lux boxes between promotors (Pl and Pr), increasing expression of the regulon
What does Lux Operon contain?
lux I, C, D , A, B, E,G
Lux I
synthesizes AI (HSL)
- lexAB code for luciferase alpha and beta subunits, the light producing enzyme, simultaneously oxidizes FMNH2 and a long chain aldehyde
- lux CDEG reduces and recycles the reactants
LuxR-HSL recruits ____ __________ to transcribe the Lux operon
RNA polymerase
LuxR is a ________
Activator
Quorum Sensing
1 - Target genes off: single cell, low [acyl HSL] -> growing aggregate, increasing [acyl HSL]
2- Target genes on: bacterial quorum, inducing [acyl HSL]
Ex: A. fischeri produces light when associated with Euprymana scolopes, but not when an individual cell is swimming in the open ocean
Microbes display ______ behavior and communicate
social
Importance of biofilms
- cooing water
- food processing
-teeth
-medical implants - drinking water
-etc
Extracellular polymeric substances (EPS)
- slimly matrix material made up of 97% water
- comprised of long-chain polysaccharides, DNA, and secreted proteins and small molecules including HSLs
- can slow diffusion of chemicals into the biofilm
- often difficult to remove the matrix even with detergents
Importance of Biolfilm formation to microbes
- Protection from antimicrobials
- some microbes transitioning to biofil growth increase expression of efflux pumps (to pump antimicrobials out) - Cooperation
- enhance nutrient access and availability
- facilitate acquisition of new traits through gene transfer - Allows microbial cells to hide from host immune system (macrophages) or hide from predators
- Facilitates phenotypic diversity of a population
One reason for resistance is that the biofilm lifestyle promotes physiological ____________
diversity
oxygen availability will affect the metabolism of a bacterium
some antibiotics only kill actively growing cells
The cells at the base of the biofilm are most likely….
dormant because of limited access to nutrients and oxygen, outside cells get all the good stuff
An increase in c-di-GMP levels (intracellular signals), all together transition the cells into a _______ state
biofilm
CtrA~P prevents cell __________
divison
- blocks DnaA from attaching to oriC
-activates flagella genes important for swarming
CtrA~P is eventually proteolyzed by ______, which then allows replication to proceed
ClpXP
How does endospore formation in B. subtilis arise?
- brought on by nutrient stress
- developmental process involves the differential regulation of 700+ genes
B. subtilis versatile responses to changing conditions
- environmental and internal signals, detected by specific sensor kinases, activates the sensor and leads t phosphorylation of transcription factors DegU ComA or Spo0A
The transcription factor Spo0A makes….
a spore (through asymmetric division)
Spo0A
- the Master Regulator
- Genes with strong activator binding sites are expressed first, others with weaker boxes require Spo0A to accumulate to high levels before they are expressed
This allows B. subtilis to explore several responses (e.g making biofilm, making toxins to kill siblings) before restoring to spore formation
Classes of genes
Transcription factors
Sigma factors
Anti-sigma factors
The Spo0A uses _____ ____________ regulation: anti-sigma factors and inactive “pro-proteins” to control the activity of alternative sigma factors
post-translational
Regulatory mechanisms to initiate sporulation
- Integration of environmental and internal conditions (availability of specific nutrients, population density, metabolic stress, DNA damage, etc)
- Kinases and phosphatases impact the activation of Spo0A pool
E. coli sigmas you need to know
sigma 70 - vegetative
sigma H and E - heat shock
sigma S - stationary phase
sigma F - flagella synthesis
B. subtilis sigmas you need to know
sigma A - vegetative (house keeping)
sigma B - stress
sigma D - chemotaxis
sigma H - early stationary phase
sigma F,E,G,K - sporulation, cell-specific
7 ECF sigmas - extra cytoplasmic function - envelop stress (from antibiotics that attack the cell envelope)
Developmental progression is driven by the sequential activation of _________ ________
sigma factors
Sigma F
?????
Signal transduction from the forespore to the mother cell activates…..
sigma E
What gets stained when studying a bacillus subtilis?
Membrane - Red
GFP-tagged protein - Green
DNA - Blue
What is used to visualize gene expression in B. subtilis?
GFP: expression is controlled by a specific promotor
The start of sporulation is triggered by …….
unfavorable conditions (starvation, desiccation, temperature, etc)
Then Spo0A~p causes expression of SpoIIE
_______ triggers differentiation of the forespore from the mother cell
Sigma F
Which an anti-sigma factor?
Sigma F
Sigma E
SpollAA
SpollAB
SpoOA
SpollAB
Which an anti anti-sigma factor?
Sigma F
Sigma E
SpollAA
SpollAB
SpoOA
SpollAA
Endospore Properties
- protein coats, outer membrane - barrier to solvents and enzymes
Cortex - special PG, low cross linking compared to cell wall,
essential for dehydration of core
Core - low water content, high Ca2+
Survival mechanisms of endospore
- Disperse!
- Prevent damage or mange and repair
Low water content- protect proteins from denaturation
SASPs (small acid soluble proteins) coat DNA - protect against depurination (removal of A and G bases
During germination - mobilize DNA repair mechanisms
