Topic 8 - Regulation Flashcards
role of DNA (4 - replication, transcription, translation, repair)
replication - DNA must be retained intact and copied to make new cells
transcription - DNA must be turned into multiple “working copies” to provide instructions for enzymes/structural proteins production
translation - RNA must be read and decoded to form the enzymes/structural proteins of cell
DNA repair - systems need to be able to deal w/ dmg
transcription in bacteria! (sigma factor details)
- sigma factors bound to RNA pol core enzyme direct it to a promoter
- transcription proceeds
- diff sigma factors can direct core RNA pol enzyme to diff genes (as needed)
rho-dep/indep transcription in bacteria
rho-dependent:
- rho protein follows RNA pol and removes it from DNA when it reaches a termination sequence
rho-independent
- RNA hairpin loop forms, causing RNA pol to dissociate from DNA (terminator loop)
translation in bacteria
- the small ribosome subunit and Shine-Dalgarno seq help align all machinery to correct starting location
multiple Shine-Dalgarno seq allow bacterial mRNA to be ________
polycistronic
polycistronic meaning
multiple separate proteins are encoded on one mRNA (common in prok)
why we need regulation?
diff env conditions
- changes in nutrients/availability
- changes in competition
permits condition-specific responses
- substrate specificity
- metabolism and transport
- sporulation
excess protein production wastes energy
key cellular enzymes are _____, AKA ____ ____
constitutive
housekeeping genes
e.g., TCA cycle, ATP synthases
constitutive vs inducible?
constitutive genes - always need to be on
inducible genes - only needed at certain times
basic control of gene expression can take place on the level of? (3)
transcription
translation
post-translation (enzyme activity)
ways to regulate protein activity (2)
- covalent modifications: may alter enzyme conformations
- allosteric regulation
allosteric meaning, details
“other site”
- activity inhibition/activation from binding of an allosteric effector molecule
- binding of a non-substrate molecule at a site away from active site
- conformation altered (inhibition - substrate no longer binds; activation - substrate is able to bind)
- allosteric inhibitor often end product of multi-step pathway
the operon
transcriptional unit with a series of structural genes and their transcriptional regulatory elements (e.g., lac operon)
where are operator, promoter, activator binding site, and structural genes in relation to each other?
which parts use positive / negative control of transcription?
order of left to right:
activator binding site (positive control), promoter, operator (negative control), structural genes
regulatory elements vs operon?
regulatory elements:
- activator binding site, promoter, operator
operon (regulatory elements + structural genes):
- activator binding site, promoter, operator, structural genes
positive control is ______ of promoter
negative control is ______ of promoter
upstream
downstream
can operons have more than one promoter?
yes, each with their own control system
negative control of transcription (2 types)
both involve a repressor protein
repression:
- inhibit transcription in response to signal
- minority of enzymes are controlled by repression
- typically affects anabolic (biosynthetic) enzymes
induction:
- DErepression of enzyme production in response to signal
- typically affects catabolic enzymes
- enzymes synthesized only when substrates available
default mode of induction-controlled site
gene is off (negative control);
co-inducer molecule removing repressor protein turns gene on
positive control of transcription
- allosteric regulator proteins activate binding of RNA pol to DNA
– activator proteins bind specifically to ACTIVATOR BINDING SITE of promoter - positively controlled promoters WEAKLY bind RNA pol
– activator protein recruits polymerase to promoter
– may cause DNA structural change
– may interact directly w/ pol
– can be close to promoter or 100s bp away
maltose catabolism in E.coli is an example of?
positive control of transcription
- maltose activator protein only binds DNA in presence of maltose
effectors (effector molecules) meaning, types/examples
collective term for molecules that affect protein production in association with allosteric protein regulators
- co-inducers or co-activator: substance that turns on enzyme production (induction, activator binding/positive)
- co-repressor: substance that binds and activates a repressor (repression)
- effectors interact with DNA-binding proteins
glucose is easier to eat than lactose, so
the lac operon is not expressed until ______________________, AKA?
until all glucose is consumed
- diauxic growth (2 growth phases; glucose easier, eat lactose later)
functions of beta-galactosidase and permease
beta-galactosidase - cuts lactose into 2
permease - brings it into cell
what structural genes does the lac operon have (3)? what proteins do they code for?
(upstream of promoter) what does lacI (lac-i) code for?
lacZ - beta-galactosidase
lacY - permease
lacA - beta-galactosidase transacetylase
lacI - LacI repressor
lac operon uses ____ regulated expression
inducible (catabolism)
- system only turned on when needed
lac operon (turned on) allows use of ____ sugar
lactose
how does the repressor protein get removed in the lac operon? (details)
- permease brings lactose into cell
- beta-galactosidase cuts lactose into 2 (producing glucose-galactose & allolactose)
- allolactose = co-inducer, lets cell know there is lactose, REMOVING REPRESSOR PROTEIN!
negative control in lac operon (repressor protein + effector molecule)?
repressor protein: LacI
- binds to operator, blocking RNA pol and inhibiting transcription
effector molecule: allolactose (co-inducer)
- induces transcription by inhibiting binding of repressor (LacI) to operator
positive control of lac operon (activator protein + effector molecule)
activator protein: cyclic AMP receptor protein; CRP
- binds and increases transcription rates when effector molecule (cAMP) present (low glucose)
cAMP (coactivator) binds to CRP, active CRP-cAMP binds to activator binding site (of promoter) (transcription proceeds)
effector molecule - induces conformational change in activator protein, which increases affinity for binding site, INCREASING RNA pol AFFINITY for Lac operon promoter
operon status?
level of lacZ, lacY, lacA transcription?
is lactose metabolized?
glucose conc: low
lactose conc: high
cAMP conc: high
operon status:
- cAMP (coactivator) bound to CRP (activator) bound to activator binding of promoter
- allolactose (inducer) bound to LacI (repressor) not bound to operator
level of lacZ, lacY, and lacA transcription: high
lactose metabolized: yes
operon status?
level of lacZ, lacY, lacA transcription?
is lactose metabolized?
glucose conc: low
lactose conc: low
cAMP conc: high
operon status:
- cAMP (coactivator) bound to CRP (activator) bound to activator binding of promoter
- NO allolactose (inducer) bound to LacI (repressor); repressor STILL bound to operator
level of lacZ, lacY, and lacA transcription: low
lactose metabolized: no
operon status?
level of lacZ, lacY, lacA transcription?
is lactose metabolized?
glucose conc: high
lactose conc: low
cAMP conc: low
operon status:
- NO cAMP (coactivator) bound to CRP (activator), NOT bound to activator binding of promoter
- NO allolactose (inducer) bound to LacI (repressor), repressor STILL bound to operator
level of lacZ, lacY, and lacA transcription: low
lactose metabolized: no
operon status?
level of lacZ, lacY, lacA transcription?
is lactose metabolized?
glucose conc: high
lactose conc: high
cAMP conc: low
operon status:
- NO cAMP (coactivator) bound to CRP (activator), NOT bound to activator binding of promoter
- allolactose (inducer) bound to LacI (repressor), not bound to operator
level of lacZ, lacY, and lacA transcription: low
lactose metabolized: no
does digesting lactose increase cell’s detection of glucose levels?
lactose makes glucose + allolactose
- however, happens inside cell
- glucose detection happens when it enters
- NO does not increase detected glucose lvl
negative control (common for anabolic operons)
- effector molecules can inhibit transcription by binding to repressor protein and enhancing its ability to bind to operator (repression)
ex. tryptophan amino acid synthesis operon
attenuation
(quenching of signal)
- interruption of transcription after initiation but before termination
- control of transcription by mRNA secondary structure (terminator loop or lack of)
- interaction between translation and transcription
– if ribosome quickly follows RNA pol, rho-independent terminator hairpin RNA loops are formed in the leader seq and pol detaches
– “stalling out” of ribosome in mRNA leader seq (i.e., not enough of that AA loaded in tRNA) allows transcription to CONTINUE
does attenuation happen in euk?
reason?
no; bacteria and archaea perform transcription and translation simultaneously in same space
E.coli tryptophan attenuation example
high levels of trp -> terminator loop forms, stops transcription of structural genes
- if region 2 is avail, it would bind to 3 or 4; ribosome stops at region 2 and blocks it
- regions 3 and 4 (if made) make a terminator loop for Rho-independent transcription STOP if high lvl of trp
low levels of trp -> terminator loop does not form, transcription continues
- region is not blocked, binds to region 3 (terminator loop NOT FORMED)
Quorum sensing
quorum - members of a group (numbers) that MUST be present in order to conduct business
- a chemical signaling system that allows microbes to communicate with each other
- regulation of gene exp based on pop density
- cells release autoinducer molecules into env as pop density increases!!
regulation of gene exp based on pop density (quorum sensing)
- positive feedback
- rapid induction (quick changes to env conditions)
- links behaviour to pop density
- coordinates expensive, additive processes
- roles in interactions w euk
bioluminescence of Aliivibrio fischeri: example of ____? details, enzyme
quorum-sensing
- Lux is a prototypical quorum-sensing system in A. fischeri
- A. fischeri lives freely or symbiotically with Hawaii bobtail squid
– 95% flushed out, then division allows squid to use again at night during hunting for camouflage
– cells only emit light (via enzyme LUCIFERASE) when in light organ of squid
Aliivibrio fischeri details
(AHL, LuxI, LuxR, lux box, luciferase)
- cells only emit light via enzyme luciferase when in squid’s light organ
- when grown to high density, cells produce lots of N-acyl-homoserine lactose (AHL - coactivator, autoinducer), which stimulates luminescence
- LuxI protein catalyzes AHL synthesis
- LuxR (activator) = a regulator transcriptional activator, interacts with AHL when it reaches high enough conc
– binds “lux box” DNA regulatory site (activator binding site) - leads to transcription of luciferase protein genes and luxI, which creates positive feedback loop, making more AHL
mechanisms controlled by quorum sensing (general) (4)
____ may play a role in _____
- motility
- conjugation
- biofilm formation
- pathogenesis
autoinducers may play a role in competition
- interruption or inhibiting a control pathway in other organisms in the env
two-component regulatory systems
- can use one protein as a sensor, another to control transcription
- allows for response to changes in env
- signal transduction (chemical communication between proteins) induced inside cell alters it to respond appropriately
two-component regulatory systems often involve:
- a sensor kinase (e.g., HPK): detects env stimulus (component 1)
- a response regulator (RR): regulates transcription (component 2)
- kinases add phosphate group to typically histidine residues (called histidine protein kinases (HPK)
- response regulators dephosphorylate (short-lived, temporary response)
two-component regulatory system general steps
- external signaling molecule binds input domain of component 1 (sensor)
- transmitter domain of component 1 becomes phosphorylated using ATP
- sensor transfers phosphate group to component 2 (response regulator)
- phosphorylated response regulator interacts w DNA of target gene + RNA pol to control transcription (+/-)
virulence of A. tumefaciens (what genes, what type of control)
- vir genes found on Ti plasmid only expressed under conditions similar to a plant wound site (crown gall tumours)
- virA/virG required for expression of the other virulence genes
- positive control
- two-component regulatory system senses sugars and phenolic compounds in low pH
chemotaxis (general)
- complex bacterial behaviour modulated by shifts in protein activity
- chemotactic bacteria sense changes in chemical gradients over time
- changes induce altered directions and duration of flagella rotation, leading to directed movement over time
how to isolate mutants using chemotaxis
- isolated using a capillary tube filled w/ nutrients
- motile wild-type microbes w/ normal chemotaxis will move into tube
- those w/ mutated chemotactic proteins will remain outside tube
regulation of chemotaxis general steps (3)
step 1: response to signal
step 2: control of flagella rotation
step 3: adaptation
chemotaxis:
step 1: response to signal
MCPs sense specific attractants and repellents
- MCP = methyl-accepting chemotaxis proteins (in all bacteria)
- initiates signal transduction (or not)
- lower sensitivity when closer to attractants/repellents
- higher sensitivity when farther away
- CheA = sensor protein
chemotaxis:
step 2: controlling flagella rotation
CheY protein
- phosphorylated by CheA-P when attracts = low
- CheY-P intiates flagellar reversal: tumbling
“Che” in CheY/CheA =?
chemotaxis
chemotaxis:
step 3: adaptation
feedback loop
- allows system to reset
- allows temporal detection of signal conc
- requires modification of MCPs by methylation
methylation alters “sensitivity” to binding chemicals
- phosphorylation (from repellent binding) -> affects CheA, CheZ, CheB (no flagellar change if no phosphorylation)
- methyl groups added to MCPs (when approaching attractants) = fully methylation -> low sensitivity
Che proteins: a two-component regulatory system
- CheA = sensor kinase, becomes phosphorylated
- CheA then phosphorylates CheY (RR)
- CheY binds to flagellar motor, changing activity
by interacting with CheW proteins, ____ of CheA is _____; meaning?
by interacting with CheW, autophosphorylation of CheA is modulated;
- attractants decrease phosphorylation
- repellents increase phosphorylation
if MCP is bound by an attractant, does CheA phosphorylate? How is CheY affected?
No
CheY is also not phosphorylated, so direction does not change
meaning of adaptation in chemotaxis
methylation of MCPs also regulates attraction during periods of very high attractant levels in a process - adaptation
- highly methylated MCPs will only respond to very high lvls of attractant
- if very high lvl aren’t maintained, phosphorylation of CheA/CheB will lead to eventual demethylation of MCP
- results in greater sensitivity to attractant, helping system reset and avoid saturation over time
regulons (meaning, 2 situations)
set of genes that are coordinated together, responding to the same regulatory systems
- catabolite repression: shutdown of several systems that use various nutrients when GLUCOSE = present
- SOS response: multigene system for wide-scale DNA repair in response to serious DNA dmg
two most important regulatory proteins for SOS response regulon are:
recA lexA
SOS response general steps (RecA/LexA)
DNA is damaged:
- RecA binds ssDNA and becomes active
- LexA is destroyed
- SOS genes expressed
RecA cleaves LexA repressor; SOS genes are induced
DNA is repaired:
- ssDNA not present
- RecA is inactive
- LexA represses SOS genes
low level of LexA production keeps SOS regulon genes repressed
alternative sigma factors
in bacteria, use of diff sigma factors directs RNA pols to certain genes
- most E. coli promoters are recognized sigma-70
sigma-54 / sigma-32 / sigma-38
sigma-54: nitrogen utilization genes regulator
sigma-32: heat shock protein gene regulator
sigma-38: general stress response gene regulator
