Chapter 16: Control of Gene Expression in Prokaryotes Flashcards
Control regions (cis-acting)
elements upstream of the operon that affect transcription, eg. promoter, operator
Trans-acting elements
diffusible product that interacts with a control region
A gene product (usually a diffusible protein or an RNA molecule) that acts to regulate the expression of a target gene.
Lactose
sugar (energy source) broken into simpler sugars galactose and glucose
Beta Galactosidase
(b-galactosidase)
Long Version
(product of lacZ gene)
breaks lactose into galactose and glucose
Much of the genetic analysis leading to the lac operon model was done by Francois Jacob and Jacques Monod in early 1960s
A bacterial enzyme, encoded by the lacZ gene, that converts lactose into galactose and glucose.
Lactose present in the extracellular environment.
The catabolic conversion of the disaccharide lactose into its monosaccharide units, galactose, and glucose.
Beta Galactosidase
b-galactosidase
(product of lacZ gene)
breaks lactose into galactose and glucose
Two other genes (lacY [lactose permease] and lacA [trans-acetylase]) are transcribed from this operon…..
the E. coli lac operon
structural genes
The structural genes of the lac operon are transcribed into a single polycistronic mRNA, which is translated simultaneously by several ribosomes into the 3 enzymes encoded by the operon.
locus
upstream gene encoding a “repressor” protein.
Shuts down lac operon when there is no lactose present.
E. coli lac operon
Lactose: sugar (energy source) broken into simpler sugars galactose and glucose
Beta Galactosidase (product of lacZ gene) breaks lactose into galactose and glucose
Two other genes (lacY [lactose permease] ad lacA [trans-acetylase]) transcribed from this operon
operon
Technical Definition
A genetic unit consisting of one or more structural genes encoding polypeptides, and an adjacent operator gene that regulates the transcriptional activity of the structural gene or genes.
structural gene
Technical Definition
A gene that encodes the amino acid sequence of a polypeptide chain.
Operon
multiple genes in one unit arranged for coordinated gene expression
cis-acting sequence
Technical Defnintion
A DNA sequence that regulates the expression of a gene located on the same chromosome.
This contrasts with a trans-acting element where regulation is under the control of a sequence on the homologous chromosome.
What enzyme breaks lactose into galactose and glucose?
Beta Galactosidase
The ______ conversion of the disaccharide lactose yields its monosaccharide units: ____, and _____.
The catabolic conversion of the disaccharide lactose into its monosaccharide units, galactose, and glucose.
The structural genes of the lac operon are transcribed into a single polycistronic _____, which is translated simultaneously by several _____ into the 3 enzymes encoded by the _____.
The structural genes of the lac operon are transcribed into a single polycistronic mRNA, which is translated simultaneously by several ribosomes into the 3 enzymes encoded by the operon.
IPTG
molecule that is able to induce lac operon
Turns on system more quickly, useful for experimental procedures
Molecule structure is similar to one-half of lactose.
The gratuitous inducer isopropylthiogalactoside (IPTG).
inducer
An effector molecule that activates transcription.
DNA binding proteins typically contain a conserved DNA-binding domain with characteristics such as:
- helix-turn-helix motif
- zinc finger
- leucine zipper
helix–turn–helix (HTH) motif
In DNA-binding proteins, the structure of a region in which a turn of four amino acids holds two helices at right angles to each other.
In a helix–turn–helix or homeodomain, 3 planes of the -helix of the protein are established, and these domains bind in the grooves of the DNA molecule.
zinc finger
A class of DNA-binding domains seen in proteins.
They have a characteristic pattern of cysteine and histidine residues that complex with zinc ions, throwing intermediate amino acid residues into a series of loops or fingers.
In a zinc finger, cysteine and histidine residues bind to a atom. This loops the amino acid chain out into a fingerlike configuration.
leucine zipper
In DNA-binding proteins, a structural motif characterized by a stretch in which every 7th amino acid residue is leucine, with adjacent regions containing positively charged amino acids.
Leucine zippers on two polypeptides may interact to form a dimer that binds to DNA.
A leucine zipper is the result of dimers from leucine residue at every other turn of the -helix in facing stretches of two polypeptide chains. When the -helical regions form a leucine zipper, the regions beyond the zipper form a Y-shaped region that grips the DNA in a scissorlike configuration.
Wild type operon with NO lactose (or IPTG) present:
Wild type repressor binds to promoter site, BLOCKS transcription, so….
no RNA —> no proteins
this is “Negative Control” of the operon.
Wild type operon WITH lactose (or IPTG) present
Repressor protein changes shape upon binding of lactose.
Proteins that change shape when binding another molecule are known as allosteric proteins.
E. coli lac operon mutation studies determine:
- Whether some parts of the lac operon are cis-acting (able to control the expression of genes ONLY when on the same piece of DNA),
- Whether some parts of the lac operon are trans-acting (able to control expression of genes on other DNA molecules,
- And what is the hierarchy of the different types of mutations?
Hierarchy of mutations
- Promoter minus (P-) mutations (disables RNA polymerase binding), supercedes all other mutations in cis: system OFF all the time
- O(c) mutations in cis supercede any I mutation: system ON all the time
- I(s) dominant over I+ is dominant over I-
promoter element
An upstream regulatory region of a gene to which RNA polymerase binds prior to the initiation of transcription.
Promoter minus (P-) mutations
disables RNA polymerase binding.
Supercedes ALL other mutations in the heirarchy in cis.
System OFF all the time!
O(c) mutations in cis
supercede any I mutation: system ON all the time
The second type of mutation in the hierarchy
I(s) mutation
is dominant over I+ is dominant over I-
allosteric proteins
proteins that change shape when binding another molecule
P-
Mutation at P site.
NO binding of RNA Polymerase possible
Promoter minus (P-) mutations supercede all other mutations in cis.
system OFF all the time
Trans mutation
produces a diffusible product, can be rescued with I+
Mutant (I-):
constitutive expression,
ON all the time
Rescue mutant experiments
Transform mutant E. coli with a plasmid (circular DNA molecule) called F’ that carries various parts of the lac operon
Creates “partial diploid” bacteria, has 2 copies of some genes, thus “partial”
Determine if we can rescue the mutant phenotype
System induced with IPTG (in place of lactose)
Do we see functional B-gal when there is NO lactose present?
NO (OFF)
Do we see functional B-gal when there I(s) lactose present?
YES (ON)
I+ is a diffusible product, so…
it can rescue operon function
I+Z+Y+
wild type
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? OFF
Permease (Y):Induced? ON
Permease (Y): Non-induced? OFF
I-Z+Y+
WILD TYPE, with regulation, NO lactose or IPTG?
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? ON
Permease (Y):Induced? ON
Permease (Y): Non-induced? ON
WILD TYPE, with regulation, NO lactose or IPTG?
I+Z-Y+/ F’I-Z+Y+
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? OFF
Permease (Y): Induced? ON
Permease (Y): Non-induced? OFF
I+ is a diffusible product, so it can rescue operon regulation
I-Z-Y+/ F’I+Z+Y-
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? OFF
Permease (Y):Induced? ON
Permease (Y): Non-induced? OFF
I+ is a diffusible product, so it can rescue operon regulation
I-Z-Y+/ F’I-Z+Y+
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? ON
Permease (Y):Induced? ON
Permease (Y): Non-induced? ON
NO diffusible I+ here
(∆I,Z,Y)/ F’I-Z+Y+
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? ON
Permease (Y):Induced? ON
Permease (Y): Non-induced? ON
I, Z, Y deletion, NO regulation from the F’
I+ dominant over _____
I-
I(s)
superrepressor mutation:
repressor cannot bind lactose; therefore, the system is OFF
Cannot be rescued by I+ because the operon will be bound by the diffusible I(s) product
I(s) Z+Y+
B-gal (Z): Induced? OFF
B-gal (Z): Non-induced? OFF
Permease (Y):Induced? OFF
Permease (Y): Non-induced? OFF
I(s) superrepressor mutation: OFF all the time
I(s) Z+Y+ / F’I+
B-gal (Z): Induced? OFF
B-gal (Z): Non-induced? OFF
Permease (Y):Induced? OFF
Permease (Y): Non-induced? OFF
I(s) superrepressor mutation cannot be rescued by diffusible I+
(because all the binding sites have I on them)
I(s) superrepressor mutation cannot be rescued by diffusible I+
because…..
all the binding sites have I on them
I(s) Z+Y+ / F’I-
B-gal (Z): Induced? OFF
B-gal (Z): Non-induced? OFF
Permease (Y):Induced? OFF
Permease (Y): Non-induced? OFF
I(s) superrepressor mutation: OFF all the time, I+ or I- on the plasmid have no effect
O(c)
constitutive cis mutant in the operator DNA.
No repressor can bind, system ON all the time
Cannot be rescued with operon linked to O+ operator
O+Z+Y+ (I+)(wt)
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? OFF
Permease (Y):Induced? ON
Permease (Y): Non-induced? OFF
O+Z+Y+/F’O+Z-Y+ (I+)
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? OFF
Permease (Y):Induced? ON
Permease (Y): Non-induced? OFF
O(c) Z+Y+ (I+)
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? ON
Permease (Y):Induced? ON
Permease (Y): Non-induced? ON
O(c) constitutive mutant, cannot bind I+
O+Z+Y-/ F’O(c) Z+Y+ (I+)
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? ON
Permease (Y):Induced? ON
Permease (Y): Non-induced? ON
The O+ here is linked to Y-, but getting Y all the time
O+Z+Y-/ F’O(c) Z-Y+ (I+)
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? OFF
Permease (Y):Induced? ON
Permease (Y): Non-induced? ON
O(c) operon ON, but no Z made
O+Z-Y+/ F’O(c) Z+Y- (I+)
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? ON
Permease (Y):Induced? ON
Permease (Y): Non-induced? OFF
O(c) ON all the time, but no Y product made, Y appears regulated
I(s) O+Z+Y-/ F’O(c) Z+Y+ (I+)
B-gal (Z): Induced? ON
B-gal (Z): Non-induced? ON
Permease (Y):Induced? ON
Permease (Y): Non-induced? ON
On all the time, I(s) cannot bind O even though it cannot bind lactose, O(c) dominant mutation
One more control on the lac operon
- End result of what we have examined is to produce glucose and galactose from lactose
- What if glucose is already present?
- Presence of glucose shuts down operon: Catabolite repression (presence of the “breakdown” product shuts down the system)
catabolite repression
The selective inactivation of an operon by a metabolic product of the enzymes encoded by the operon.
example of catabolite repression:
Glucose
In the presence of glucose, cAMP levels decrease, CAP–cAMP complexes are not formed, and transcription is not stimulated.
Glucose present —->
- –> inhibits adenyl cyclase
- –> don’t get cAMP
- —> cAMP doesn’t bind CAP
- –> CAP cant bind CAP binding site efficiently
- –> RNA polymerase doesn’t bind
- –>Transcription Diminished
- –> Translation Diminished
One more control on the lac operon
- End result of what we have examined is to produce glucose and galactose from lactose
- What if glucose NOT present?
Glucose NOT present
- -> adenyl cyclase works
- -> Get cAMP
- —> cAMP binds CAP
- –> CAP-cAMP complex efficiently binds CAP binding site
Glucose NOT present
Example of “Positive Control”
Glucose NOT present
- -> adenyl cyclase works
- -> Get cAMP
- —> cAMP binds CAP
- –> CAP-cAMP complex efficiently binds CAP binding site
- –> RNA polymerase binds
- –> Transcription Enhanced
- –> Translation Enhanced
“Positive Control” of operon by CAP-cAMP binding
lac operon regulation
- Various control systems we have examined reveals high degree of specificity in the genetic regulation of this operon
- End result is efficient energy use and production of lactose metabolism enzymes only when needed
Which term is used to describe enzymes that are produced only when specific substrates are present in the environment?
inducible
This term best describes enzymes that are produced only when specific substrates are present in the environment to induce their production.
True or False?
Lactose is an inducer of the lac operon.
True
Lactose indirectly induces or stimulates the transcription of genes involved in its metabolism.
What would be the effect of a mutation in the lacI gene that prevented the repressor from binding to lactose?
The lac Z, Y, and A genes would not be expressed.
If lactose could not bind to the repressor, the repressor would stay bound to the operator and repress the transcription of the lac Z, Y, and A genes.
What is the role of glucose in catabolite repression?
It decreases the levels of cAMP in the cell, repressing transcription from the lac operon.
Glucose decreases the levels of cAMP in the cell, preventing formation of the CAP–cAMP complexes necessary for the stimulation of transcription from the lac operon.
Regulation of Gene Expression: Prokaryotes
Organisms have refined mechanisms to control the transcription of genes and optimize the use of available resources.
Gene regulation in bacteria:
the expression of genes involved in lactose metabolism is modulated according to the metabolic needs of the organism.
operator region
In bacterial DNA, a region that interacts with a specific repressor protein to regulate the expression of an adjacent gene or gene set.
The lac operon is a gene cluster composed of a promoter-operator region and 3 structural genes that function in lactose metabolism.
These genes are coordinately regulated by a negative control system.
In the _____ of lactose, a repressor protein produced by the ____ gene binds to the operator region of the operon and represses transcription of the structural genes.
In the absence of lactose, a repressor protein produced by the lacI gene binds to the operator region of the operon and represses transcription of the structural genes.
In the absence of _____ , a repressor protein produced by the lacI gene binds to the _____ region of the operon and _____ transcription of the structural genes.
In the absence of lactose, a repressor protein produced by the lacI gene binds to the operator region of the operon and represses transcription of the structural genes.
If lactose is present, the ____ is prevented from binding to the operator and transcription ____.
If lactose is present, the repressor is prevented from binding to the operator and transcription proceeds.
The importance of the ____ and _____ in the regulation of the lac operon has been confirmed by experiments with mutants in which the operon is either constitutively active or inactive.
The importance of the repressor and operator in the regulation of the lac operon has been confirmed by experiments with mutants in which the operon is either constitutively active or inactive.
The lac operon is also subject to positive control through _____, which binds to a specific region of the promoter and then stimulates the binding of RNA polymerase.
The lac operon is also subject to positive control through the catabolite-activating protein (CAP), which binds to a specific region of the promoter and then stimulates the binding of RNA polymerase.
The lac operon is also subject to ____ control through the catabolite-activating protein (CAP), which binds to a specific region of the ____ and then stimulates the binding of RNA polymerase.
The lac operon is also subject to positive control through the catabolite-activating protein (CAP), which binds to a specific region of the promoter and then stimulates the binding of RNA polymerase.
Polymerase binding is efficient only in the presence of a ____ at the CAP promoter site.
Polymerase binding is efficient only in the presence of a bound CAP at the CAP promoter site.
CAP cannot bind RNA polymerase unless CAP is first bound to ____, and when ____ is in the cell, the level of cAMP is low.
CAP cannot bind RNA polymerase unless CAP is first bound to cAMP, and when glucose is in the cell, the level of cAMP is low.
In the presence of ____, the absence of ____ greatly reduces transcription from the lac operon.
In the presence of glucose, the absence of cAMP greatly reduces transcription from the lac operon.
This mechanism is known as catabolite repression and is energetically efficient because bacteria favor the use of ____ over ____.
This mechanism is known as catabolite repression and is energetically efficient because bacteria favor the use of glucose over lactose.
Which term refers to a contiguous genetic complex that is under coordinate control?
operon
In the lactose operon, the product of structural gene lacZ is capable of ________.
splitting the β-linkage of lactose
Constitutive mutations may occur in various components of the lac operon.
Mutations in which two genes are constitutive?
lac__ and lac ___.
lacI- and lacO(c)
Which of the following mutations could lead to constitutive expression of the genes of the lac operon?
A mutation in the lac-Z gene.
A mutation in the operator sequence.
A super repressor mutation.
A mutation in the lac-Y gene.
A mutation in the operator sequence
Such a mutation could prevent binding of the repressor, allowing expression under all conditions.
Which of the following best describes the biological role of the lac operon?
It ensures that a cell dedicates resources to the production of enzymes involved in lactose metabolism only when lactose is available in the environment.
The cell expends energy to produce the proteins necessary for lactose metabolism only when lactose is present.
True or False?
The placement of the operator sequence between the promotor and the structural genes is critical to the proper function of the lac operon.
True
When the repressor binds to the operator, RNA polymerase cannot transcribe the structural genes.
Which of the following is an example of positive control in operons?
the action of the catabolite activator protein (CAP) in the lac operon
CAP binds to its binding site in the lac operon and stimulates transcription.
What is the role of glucose in catabolite repression?
It decreases the levels of cAMP in the cell, repressing transcription from the lac operon.
Glucose decreases the levels of cAMP in the cell, preventing formation of the CAP-cAMP complexes necessary for the stimulation of transcription from the lac operon.
Under the system of genetic control of the tryptophan operon, ________.
when there is no tryptophan in the medium, transcription of the trp operon occurs at high levels.
Regarding regulation of the tryptophan operon, which type of regulatory molecule might one appropriately call the amino acid tryptophan?
corepressor
The tryptophan operon is typically characterized what two terms?
negative control and repressible
Which of the following occurs as a result of an abundance of tryptophan in E. coli?
The 5 trp genes (TrpA – TrpE) are not transcribed.
When trp is abundant, the genes involved in tryptophan synthesis are negatively regulated at the level of transcription.
Which of the following features of the trp operon is likely least essential to the process of attenuation?
The order of the structural genes, E, D, C, B, A
Because each gene encodes an enzyme involved in the trp synthetic pathway, the order in which the genes occur is likely not important in terms of the attenuation process.
True or False?
Attenuator systems such as the one described for regulation of tryptophan synthesis would be just as likely to occur in eukaryotes as in prokaryotes.
FALSE
Regulation by attenuation requires that translation of a given transcript can begin before transcription is completed. This is not possible in eukaryotes, as the two processes are spatially separated by the nuclear membrane.
What is the function of the hairpin loop in the mRNA transcript produced by the trp operon?
The hairpin causes transcription to stop.
When the leader peptide sequence is transcribed, it gives rise to an RNA structure that folds back on itself and forms a hairpin loop. The loop is a terminator that causes RNA polymerase to cease transcription, dissociate from the DNA, and release the truncated mRNA.
Which of the following statements about the mechanism of attenuation is true?
When tryptophan levels are low, transcription is initiated and continues through the leader region into the structural genes, resulting in the production of a polycistronic mRNA and, ultimately, the enzymes required for the biosynthesis of tryptophan.
This describes the regulation of the trp operon by attenuation.
When referring to attenuation in regulation of the tryptophan operon, it would be safe to say that when high levels of tryptophan are available to the organism, ________.
transcriptional termination is likely
A stretch of DNA consisting of an operator, a promoter, and genes for a related set f proteins, usually making up an entire metabolic pathway.
Operon
Are usually arranged sequentially after the promoter…
The genes of an operan
A specific nucleotide sequence in DNA that binds RNA polymerase, positioning it to start transcribing RNA at the appropriate place.
Promoter
Codes for a protein, such as a repressor, that controls the transcription of another gene or group of genes
Regulatory Gene
Regulatory Proteins bind to the ___ to control expression of the operon.
Operator
A protein that inhibits gene transcription. In prokaryotes, this protein binds to DNA in or near the promoter.
Repressor
A specific small molecule that binds to a bacterial regulatory protein and changes its shape so that it cannot bind to an operator, thus switching an operon on.
Inducer
An operon is made up of a ___ and ____.
An operon is made up of a promoter and the genes of the operon.
The promoter, which includes an operator, is the stretch of DNA where _____ binds.
The promoter, which includes an operator, is the stretch of DNA where RNA polymerase binds.
Regulatory proteins bind to ____.
Regulatory proteins bind to the operator.
The genes of the operon code for ____.
The genes of the operon code for a related set of proteins.
A regulatory gene located away from the operon codes for a protein that _____ .
A regulatory gene located away from the operon codes for a protein that controls the operon.
when operon is not transcribed …
trp operon: tryptophan present
lac operon: lactose absent
When operon is transcribed, but not sped up by the positive control….
lac operon: lactose present, glucose present
trp operon: tryptophan absent
When operon is transcribed quickly through postive control…
lac operon: lactose present, glucose absent
The ____ operon is regulated through negative control only.
The trp operon is regulated through negative control only.
The ____ operon is regulated through both negative control and positive control.\
The lac operon is regulated through both negative control and positive control.
The trp operon is regulated through negative control only.
When tryptophan is present, the ____ are not transcribed.
The trp operon is regulated through negative control only. When tryptophan is present, the operon genes are not transcribed.
Negative Control Operon Example:
When lactose is absent, the repressor protein is active, and transcription is turned off.
When lactose is present, the repressor protein is inactivated, and transcription is turned on.
Positive Control Operon Example:
When glucose is absent, another regulatory protein (CAP) binds to the promoter of the lac operon, increasing the rate of transcription if lactose is present.
The locations of numerous lacI− and lacIS mutations have been determined within the DNA sequence of the lacI gene. Among these, lacI− mutations were found to occur in the 5’-upstream region of the gene, while lacIS mutations were found to occur farther downstream in the gene. Are the locations of the two types of mutations within the gene consistent with what is known about the function of the repressor that is the product of the lacI gene?
The mutations described are consistent with the structure of the lac repressor.
