Module 6: Reg. of Gene Expression (Expression and Operons) Flashcards
Gene Expression
Process by which information from a gene is used in the synthesis of a functional gene product (typically proteins)
Not ALL gene products…
Not ALL gene products are needed ALL the time!
Controlling gene expression is used to control…
what gene products are present within a cell
Gene expression control occurs at what overall levels?
3 levels:
1) Transcriptional Level
2) Translational Level
3) Post-Translational Level
Transcriptional level of regulation is generally determined by…
Whether RNA polymerase binds to a gene promoter or not (initiating transcription)
Translational level of regulation is generally determined by…
Whether mRNA “makes it” to/binds to ribosome to initiate translation
–> Can be impacted by mRNA secondary structure
At the translational level, mRNA secondary structure can impact:
1) Susceptibility of mRNA to enzymatic degradation (by RNAses)
2) Inhibition of ribosomal binding
Post-Translational level of regulation is generally determined by…
Modifications made to a formed protein to either ACTIVATE or DEGRADE it
The __________ regulation occurs the …..
The sooner regulation occurs the less energy and resources are wasted
Controlling gene expression is what kind of a process?
CONSERVATIVE PROCESS
–> Conserves energy and resources
–> Why make a protein just to degrade/deactivate it when you can prevent its formation in the first place?
What are the two classifications of gene expression?
1) Constitutive expression
2) Inducible expression
Constitutive Expression
The constant expression of genes that encode for products ALWAYS needed by a cell for essential processes
–> Genes are constantly “on”; constant gene product generation
What specific pathways involve constitutively expressed genes (3)?
Genes involved in:
1) Glycolysis
2) Transcription
3) Translation
Inducible Expression
The SELECTIVE expression of genes that encode for products needed ONLY under certain conditions’
–> Genes are “off” and must be “turned on”; their expression is induced
Gene products of inducibly expressed genes are…
ONLY needed according to the presence of a specific substrate
Classifications of enzymatic control/regulation within cells (2):
2 Classifications:
1) Control of enzymatic ACTIVITY
2) Control of enzymatic PRODUCTION
How can activity of enzymes be regulated?
By altering ENZYMATIC STRUCTURE to alter the binding properties of an enzyme
Can occur in one of 2 ways:
1) Binding of inhibitors
2) Covalent modification
What are the two methods of regulating enzyme activity?
1) Binding of inhibitors
2) Covalent modification
What are the types of regulation of enzymatic activity by inhibitor binding?
1) Competitive Inhibition
2) Allosteric Inhibition
Competitive Inhibition
When an inhibitor molecule binds to the ACTIVE SITE of an enzyme, blocking the substrate from binding
(Inhibitor and substrate “compete” for the active site)
Why is competitive inhibition competitive?
Because the inhibitor and substrate “compete” for binding of the active site!
Allosteric Inhibition
An inhibitor molecule that binds to a site on an enzyme OTHER than the active site (allosteric site)
Binding of an inhibitor to an allosteric site causes…
conformational change to the enzyme so that the substrate can no longer bind to the active site!
Allosteric inhibitors are typically…
The PRODUCT of a multistep pathway that the enzyme is involved in
What is the technical definition of allosteric inhibition?
Inhibition of enzymatic activity by an effector molecule that binds REVERSIBLY to an enzyme’s allosteric site and is not changed in the reaction
In allosteric inhibition, the binding of the inhibitor is…
REVERSIBLE
What is allosteric inhibition AKA (in some cases)
Feedback Inhibition
Covalent Modification
Regulation of enzyme action/activity by the addition of a chemical entity (to activate or deactivate)
What are some examples of covalent modification?
1) Phosphorylation (addition of PO42-)
2) Acetylation (addition of acetyl grp: CH3C=O)
3) Methylation (addition of CH3)
4) Glycosylation (addition of carbohydrate grp)
In what context is modification of enzymatic activity great for?
RAPID RESPONSE to some change
Modification of enzymatic activity is NOT ________________ because…
Modification of enzymatic activity is NOT the most efficient method of controlling metabolic pathways
BECAUSE
It is energy expensive
–> The energy and resources to make the uncessary enzymes is already “spent”
Modifying enzymatic activity is NOT a…
NOT a conservative process
How is the production of enzymes mainly regulated?
The production of enzymes is mainly regulated via initiation of transcription
(by modulating the ability of RNA polymerase to bind a promoter)
How does RNA polymerase bind to promoters?
Via complementary base pairing
What determines the “strength” of a promoter?
The binding affinity of RNA polymerase to the promoter
What determines binding affinity of RNA polymerase to a promoter?
The presence of specific recognition sequences within the promoter
–>Specifically, CONSENSUS SEQUENCES; RNA polym. recognizes and binds to specific consensus sequences on promoters!
Consensus Sequence
DNA sequence that is most commonly found at a particular position in a set of related DNA sequences (Ex: promoters)
What molecules can control RNA polymerase binding affinity?
1) Regulatory Proteins
2) DNA binding proteins
Regulatory proteins (for transcription) are either…
ACTIVATORS = increase binding affinity of RNA polymerase to promoter (increasing transcription)
or
REPRESSORS = decrease binding affinity of RNA polymerase to promoter (typically by obstructing binding altogether) (decreasing transcription)
What is the most conservative regulation mechanism?
Transcriptional regulation (Saves the most energy and resources)
Any molecule or alterations that _____________ will alter the amount of transcription that occurs
Any molecule or alterations that impact the affinity of RNA polym. to a given promoter will alter the amount of transcription that occurs
In protein synthesis, it is often advantageous for a cell to…
(especially for products of the same pathway)
COORDINATELY regulate the production of SEVERAL proteins!
–> Especially those involved in the same pathways!
Coordinated regulation of protein synthesis allows for…
Proper activation/deactivation of a given pathway!
Operon
A transcriptional unit consisting of a series of consecutive structural genes (that are transcribed from a SINGLE shared promoter) and their regulatory elements
What are the 2 main components of an operon?
1) Structural genes
2) Transcriptional regulatory elements
Eukaryotic vs Prokaryotic Operon
Eukaryotic = MONOCISTRONIC
Prokaryotic = POLYCISTRONIC
Mono- vs Poly- cistronic
Monocistronic = mRNA transcript encodes for ONE protein
(1 RNA = 1 protein)
Polycistronic = mRNA transcript encodes for 2 or MORE proteins
(1 RNA = 2+ proteins)
Typically, the products of ONE operon…
…this facilitates…
participate in a SINGLE biochemical pathway
== simultaneous transcription facilitates efficient operation of that pathway!
lac Operon
(Source, purpose, genes)
In E.coli
–> Purpose = Transport and catabolism of lactose
–> Has 3 structural genes (lacZ, Y, A)
trp Operon
In E.coli
–> Purpose = biosynthesis of tryptophan
–> Has 5 structural genes
virB Operon
In Agrobacterium
–> Purpose = contains virulence genes involved in DNA transfer from bacteria to plant
–> Has 11 structural genes
S10 Operon
Conserved throughout all bacteria!
–> Purpose = Contains ribosomal structural genes
–> Has 11 structural genes
S10 = “almost 10” = 11 genes
nfiHDK Operon
In Klebsiella pneumoniae
–> Purpose = Production of nitrogenase enzyme involved in nitrogen fixation
–> Has 3 structural genes
Think “nfi” = nitrogen fixation + HDK = 3 letters, 3 genes
What are the components of the lac operon?
1) Promoter
2) Operator
3) three structural genes: lacZ, lacY, lacA
4) Regulatory proteins (like lacI)
What are the products of the lac operon structural genes?
lacZ = Encodes for B-Galactosidase production
lacY = Encodes for permease production
lacA = Encodes for B-galactosidase transacetylase
B-galactosidase function
Enzymatic breakdown (catabolism) of lactose
Permease function
Facilitates the uptake of lactose into the cell
B-galactosidase transacetylase function
UNKNOWN
Promoter
Site of DNA to which RNA polymerase binds
–> Directs the initiation of transcription
Operator
DNA sequence to which regulatory proteins can bind
What is the effect of regulatory proteins that bind to operators?
Their binding modulates the ability of RNA polymerase to bind to the promoter of an operon and this influences initiation of transcription
The genes encoding for regulatory proteins can be… AND they can…
found FAR or CLOSE to the operon/s they regulate AND they might regulate MORE than one operon/target genes!
In operons, regulatory proteins can either:
1) Facilitate transcription
2) Inhibit transcription
What are the two types of control in operon systems?
Negative (-) Control = Involves inhibition of an operon
Positive (+) control = Involves activation of an operon
Negative Control (in operons)
Refers to regulatory mechanisms involving REPRESSORS (regulatory molecules that decrease transcription)
Positive Control (in operons)
Regulation of transcription in which regulatory molecules INCREASE transcription (involves ACTIVATORS)
What is a repressor?
A regulatory protein that BLOCKS transcription
What do repressors bind to?
And HOW?
Repressors bind to operators
They bind as a DIMER to the operator!
Effectors
Small molecule that binds to activator or repressor proteins, modifying their gene regulation activity.
What are the types of effectors?
1) Inducers (includes co-activators)
2) Corepressors
Inducers
Effectors that increase transcription by either:
1) ENABLING an activator (coactivator)
2) DISABLING a repressor
In negative control, what do inducers do?
Bind to repressors and inhibit their binding to an operator
In positive control, what do inducers do?
Inducers act as CO-ACTIVATORS –> bind to activators to enable their activity!
Co-repressors
Effectors that enhance binding of repressors to an operator
Negative control:
Inducers vs corepressors
BOTH bind to repressors
Inducer bound to repressor = Repressor binding to operator is INHIBITED
Co-repressor bound to repressor = Repressor binding to operator is ENHANCED/ENABLED
Inducers ________ to occur whereas corepressors ___________
Inducers ALLOW TRANSCRIPTION to occur
whereas corepressors STOP TRANSCRIPTION occurring
What molecules are often effectors?
Effectors are often the intermediate products of a given metabolic pathway
Effectors binding to regulatory proteins (repressors/activators) is a form of…
Allosteric Regulation
What is a major difference between the lac and trp operons?
lac operon = INDUCIBLE operon (native state = OFF)
trp operon = REPRESSIBLE operon (native state = ON)
lacI gene
A gene that encodes for the regulatory protein LacI which is a repressor for the lac operon
–> the lacI gene is NOT a part of the lac operon!
What kind of expression does the lacI gene exhibit?
Constitutive expression; it is ALWAYS producing the LacI repressor!
When lactose is NOT present…
the LacI repressor is bound to the operator = NO transcription of the lac operon
What is the negative control process in the lac operon?
1) lacI constitutively expresses/produces LacI repressor
2) Presence of lactose = small amount of breakdown leading to allolactose production
3) Allolactose acts as an INDUCER to the LacI repressor –> Binds to the repressor, causing a conformational change which inhibits its binding to the operator
4) Operator is available so RNA polymerase is free to bind and transcription of the lac structural genes can occur
5) Translation of the lac operon transcript results in proteins involved in lactose catabolism
6) lactose is broken down
7) absence of lactose SHUTS DOWN the catabolism
What are the possible products of B-gal. breakdown of lactose?
B-gal. breaks lactose down in two potential pathways:
1) lactose –> glucose and galactose
2) lactose –> ALLOLACTOSE
What is allolactose? What is its role in the lac operon?
Allolactose = isomer of lactose
–> Acts as an inducer for the LacI repressor
What does negative control do in lac operon?
(-) control SHUTS DOWN the catabolic pathway of lactose breakdown in the absence of lactose
What is the negative control process in the trp operon?
1) trpR is constitutively expressed = INACTIVE trpR repressor is constantly produced
2) In the presence of high trp concentration, trp binds to the trpR repressor as a co-repressor = ACTIVATES repressor!
3) The activated repressor binds to the operator of the trp operon
4) Binding of RNA polym. and therefore initiation of transcription is blocked
5) No production of tryptophan
think trpR= Repressor
What does negative control do in the trp operon?
(-) control SHUTS DOWN the anabolic pathway of tryptophan biosynthesis in the presence of trp
What does trp acts as within the trp operon system?
trp = co-repressor for the trp repressor!
How is allolactose produced in the first place if the lac operon is “off”?
The lac operon is a LEAKY SYSTEM = the lac operon is never actually fully turned off
–> LOW levels of permease and B-gal are always being produced
AND
There may be some permease and B-gal leftover from previous expressions!
What is the leaky system of the lac operon?
The lac operon is never completely off
–> Always a low level of permease and B-gal production occurring even in the absence of lactose
–> Allows for SOME lactose to enter the cell and catabolize to allolactose to then begin a larger-scale induction of lactose catabolism!
Positive control involves what kind of regulatory molecules?
Activators
Activators
Molecules that increase the affinity of RNA polymerase to a promoter (that otherwise do not bind very strongly)
Activator Binding Site
DNA sequence that binds an activator molecule
A conformational change in an activator can impact…
the activator’s ability to bind to an activator binding site
Positive control systems can be shut down by (2):
1) An inhibitor that removes an activator from the activator binding site
2) Removal of the co-activator needed to activate the activator
What is the positive control process in the lac operon?
When glucose is LOW and lactose is present:
1) Allolactose is produced, blocking the repressor from binding the operator
2) CRP activator binds to available cAMP co-activator (due to low glucose)
3) activated CRP-cAMP complex binds to the activator binding site on the DNA
4) Binding of the activator triggers RNA polymerase to bind
5) Transcription of the operon begins
Why is the presence of lactose NOT enough to trigger transcription of the lac operon?
Because RNA polymerase does not have sufficient binding affinity to attach to the promoter of the lac operon by itself!
So even though the promoter site is OPEN and not blocked by repressor on the operator, RNA polymerase requires HELP to bind!
What activator is used in the lac operon?
CRP = cAMP receptor protein
CRP
AKA?
cAMP receptor protein
AKA. CAP = catabolite activator protein
What does CRP need to function?
cAMP co-activator
–> CRP is not active by itself! It becomes active by complexing with cAMP
The lac operon is under what kind of control (altogether)?
DUAL CONTROL
–> Has both negative and positive control systems
What are the determining factors for negative and positive control in the lac operon?
(-) Control = Determined by presence of lactose
(+) Control = Determined by absence of glucose
What is the benefit of the dual control of the lac operon?
It allows for the best control when MULTIPLE sugars are present!
–> Allows for the preference of glucose while keeping cell prepared to use lactose if needed
How does glucose concentration impact cAMP concentration?
As glucose concentration increases, cAMP concentration decreases
High Glucose = LOW cAMP
Low Glucose = HIGH cAMP
How does glucose impact the activation of the lac operon?
Glucose impacts how much cAMP is available for binding to the CRP activator
= Determines how much of the active CRP-cAMP complex forms
== This directly determines how much the lac operon can be activated (IF lactose levels are sufficient as well)
What is the preferred carbon source of E.coli?
WHY?
GLUCOSE is preferred!
Because it can enter glycolysis directly!
Lactose on the other hand must be catabolized before it can enter glycolysis = less efficient
(+) control system allows for glucose…
To be used FIRST even if lactose is present!
State of the lac operon when:
Low glucose, High lactose
Low Glu. = High cAMP
High Lac. = High allolactose
The operon:
Operator = OPEN –> Allolactose bound to the repressor
ABS = CRP-cAMP activator bound
–> RNA Polymerase binds the promoter and transcription of 3 structural genes occurs! = lactose is metabolized!
State of the lac operon when:
Low glucose, Low lactose
Low glu. = High cAMP
Low lac. = low allolactose
The operon:
Operator = BLOCKED –> Allolactose NOT bound to the repressor
ABS = CRP-cAMP activator bound
–> RNA polymerase CANNOT bind to the promoter due to the repressor on the operator
–> Transcription is NOT initiated = NO lactose metabolism
State of the lac operon when:
High glucose, High lactose
High Glu. = Low cAMP
High Lac. = High allolactose
The operon:
Operator = OPEN –> Allolactose is bound to the repressor
ABS = NOT BOUND –> CRP is inactive due to lack of cAMP to complex with
–> RNA polymerase CANNOT binds to the promoter due to the lack of the activator complex; promoter is too weak on its own
–> Transcription is NOT initiated = NO lactose metabolism
State of the lac operon when:
High glucose, Low lactose
High Glu. = Low cAMP
Low Lac. = Low allolactose
The Operon:
Operator = BLOCKED –> Allolactose NOT bound to the repressor
ABS = NOT BOUND –> CRP is inactive due to lack of cAMP to complex with
–> RNA polymerase CANNOT bind to the promoter due to blocking at the operator AND lack of the activator complex
–> Transcription is NOT initiated = NO lactose metabolism
Lactose is only metabolized when…
Glucose has been exhausted and lactose is present!
(LOW glucose and HIGH lactose conditions)
What is the growth curve of E.coli grown in medium with BOTH lactose and glucose?
DIAUXIC growth curve (has 2 phases)
What is the diauxic growth curve of E.coli?
A 2 phase growth:
First phase = more rapid; due to metabolism of glucose
Lag Period = glucose exhausted; cell begins transcribing and translating the lac operon
Second phase = slower growth; due to metabolism of lactose
What causes the lag in growth in the diauxic growth curve of E.coli?
Once glucose is depleted, there is a lag in growth due to the cell needing some time to transcribe and translate the lac operon BEFORE lactose can be massively uptaken or metabolized
Why is there a difference in growth rate in the 2 growth phases of the diauxic curve?
Because the phases correspond to the metabolism of different carbon sources
The first phase has a higher growth rate due to the metabolism of glucose which is much more efficient
The second phase has a lower growth rate due to the use of lactose which is not as efficient
In the discovery of the lac operon, what mutants were created?
From WT strain:
1) Z mutants
2) Y mutants
3) I- mutants
4) O- mutants
Z mutant
Did NOT have B-gal. activity
–> Was due to mutation in the lacZ gene
Y mutant
Had B-gal. activity BUT still couldn’t metabolize lactose
–> Was due to mutation in the lacY gene (preventing formation of permease and therefore preventing uptake of lactose)
I- mutant
Constitutively expressed B-gal. EVEN in the ABSENCE of lactose!
–> Had mutated lacI gene = no repressor was being produced so the operator was in a permanent “on” state
How was it determined that the I- mutants lacked proper lacI gene?
Scientists treated I- mutants with plasmid containing WT alleles of the lac operon (I+ plasmid):
–> Upon treatment, the constitutive phenotype of the I- mutant was complemented = operon became inducible again!
–> Suggested that some diffusable factor from the I+ plasmid was responsible for repressing the lac operon
Why was it important that scientists observed the lacI repressor factor (unknown at the time) to be diffusable?
Because identifying that the repressor was diffusable led to its identification as a protein!
–> Since it was diffusable, it was most likely a gene product rather than a gene itself!
What happened to the I- mutant upon treatment with the I+ plasmid?
1) The I+ plasmid with functional lacI gene began producing LacI repressor
2) LacI repressor bound to the I+ plasmid operator AND diffused over to the I- lac operator and bound to it as well
3) = Repression of the lac operons (both in the I- and I+ DNA) that caused the loss of B-gal. activity within the cells
What mutants were used to determine HOW the LacI repressor worked?
O- mutants
Had constitutive expression of the lac operon (B-gal.) activity BUT could NOT be complemented by the WT plasmid
–> Had a mutation in the operator = repressor could not bind to it
How was it determined that the O- mutants lacked proper operator?
By treating with the WT Plasmid and observing no complementation of the constitutive phenotype
== Suggested that the mutation was NOT in the gene encoding for the repressor BUT instead in the DNA sequence to which the repressor binds!
What happened to the IO mutant upon treatment with the WT plasmid?
1) WT plasmid AND the O- mutant produced LacI repressor
2) LacI repressor bound to the plasmid lac operator BUT could NOT bind to the O- chromosomal lac operator
3) B-gal synthesis (lac operon activity) was blocked in the plasmid BUT it was NOT blocked in the chromosomal lac operon
= B-gal still produced even though repressor was being made and was present within the cells
What did the I- and O- mutants lead to the discovery of?
I- mutants = led to discovery of LacI repressor
O- mutants = led to discovery of the OPERATOR
How was the promoter of the lac operon discovered?
By studying mutants that NEVER expressed BOTH LacZ and LacY
