Lac operon in E. coli
controls breakdown of lactose
regulatory gene produces active repressor [bind operator] and block RNA pol.
REGULATED AS A SINGLE UNIT.
****When lactose is available, lactose binds repressor and inactivates it => RNA pol can now transcribe.
*****NO lactose---DO NOT need enzymes to metabolize lactose
******Enzymes for lactose metabolism is INDUCIBLE. Lactose induces the operon.
inducible system
An operon that requires an inducer to remove a repressor protein from the operator site to begin transcription of the relevant gene;
also called a positive control system.
INDUCIBLE system is OFF until it turns on*******
cis-acting elements
regulatory DNA sequences involved in eukaryotic transcription initiation
can only regulate genes located on same chromosome
Genetic dissection experiments
mutations in different genes and use of partial diploids
tryptophan operon
repressible SYSTEM
when trp is absent, the repressor is inactive-----
when trp is present and there is enough of it, the trp binds to the repressor and activates it to stop producing
Prokaryote control of gene expression
-inducible system
-cis acting elements
Eukaryote control of gene expression
-generally lack operons
-various points of regulation
-transcriptional regulation
Transcriptional regulation
The mechanisms that collectively regulate whether or not transcription occurs.
-Chromatin remodeling
-epigenetics [histone acetylation, DNA methylation]
-Promoters
-Enhancers
-Insulators
Chromatin remodeling
A mechanism for epigenetic gene regulation by the alteration of chromatin structure.
Acetylation of histones: chromatin loosens or opens for transcription so the rate for transcription increases.
Deacetylation: MORE compaction and less transcription [decreased expression]
Histone acetylation
the attachment of acetyl groups [-COCH3] to tails of histone proteins
DISRUPTS chromatin structure--the chromatin becomes less compact, and the DNA is accessible for transcription
DNA methylation
The addition of methyl groups [—CH3] to bases of DNA after DNA synthesis
Associated with decreased gene expression
Methylation occurs most often on cytosine of CG doublets in DNA.**** [C adjacent to G can get methylated]
Methylation can repress transcription by binding to transcription factors of DNA.
X chromosomes in female cells are heavily methylated. [inactive X]
Many cancers DNA hypomethylation ["active" cells]
Promoters
specific region of a gene where RNA polymerase can bind and begin transcription
Recognized by transcription factors [TFs] subsequently recognized by RNA pol:
Core Promoter element: TATA box [25-30]
Proximal promoter elements: CCAAT box [about 70-80]
GC box [110]
Cis acting sequences
Located on same chromosome as gene that it regulates; ONLY regulates genes they are adjacent to.******
DNA sequences that trans-acting factors bind to
NOT involved in direct binding of RNA polymerase, but interact with TFs--possibly by altering chromatin structure and facilitate binding of RNA polymerase, or may increase the concentration of TFs near promoter [complex sex of interactions]
Required for accurate regulated transcription of genes:
1. Promoters/Promoter Proximal Elements
2. Enhancers
3. Silencers
Trans-acting factors [transcription factors]
-Recognize cis acting elements of different structural genes
-can impact regulation of more than 1 copy of a gene OR more than 1 gene.
Insulators
block or insulate the effects of enhancers in a position dependent manner
Examples of transcriptional regulation
Hormone induced response
Hormone picked up by cell, binds to receptor and acts as Transcription Factor
Recognizes cis-actining HRE DNA sequence, increases transcription of a certain set of genes
ex: Steroid hormones [glucocorticoid] and metallothioneine IIA gene [protects cells from toxic effects of heavy metals [ex: zinc]
Insulator binding proteins
When transcription factors act at a distance, the insulators [citerm-20s elements] block transcription factors from interacting with the wrong gene
Prevents transcription factor from binding to the wrong gene
Activators
up regulate;
BIND to enhancer
Repressors
down regulate;
BIND to silencer
***Repressor gene is NOT part of operon and is expressed separately; It is a regulatory protein that has its own promoter sequence.
Enhancers
located on either side of gene, some distance from gene, or even within gene
Important in reaching maximum level of transcription
Silencers
[down regulate]: repress the level of transcription initiation
Coactivator
Interact with activator, alter chromatin structure forming enhancesosome
Other types of regulation
Posttranscriptional: Alternative splicing, RNA interference
Alternative splicing
Post-translational RNA modification process in which some exons are removed or joined in various combinations.
Intro to control of gene expression
Not all genes are expressed at all times in all situations.
Many prokaryotic gene products are present continuously at low levels; these can increase as needed.
In multicellular eukaryotes, differential gene expression is also essential and is at the heart of embryonic development and maintenance of the adult state.
Levels of gene regulation
transcriptional, post-transcriptional, translational, post-translational
Bacteria and Gene expression
Bacteria respond metabolically to changes in their environment [note - single-cell organisms]. [MAINLY respond to environment; if environment changes, genes turn on or off]
Bacteria regulate gene expression to synthesize products needed for a variety of normal cellular activity.
-DNA replication
-Recombination
-Repair
-Cell division.
Structural genes
Protein-encoding DNA sequences that function in metabolism, biosynthesis, or cell structure
**Important in maintenance
Genes coding for the primary structure of an enzyme
Regulatory gene
Protein- or RNA-encoding DNA sequences that regulates structural genes [trans-acting]*******
Help to control the expression of structural genes of the operon by increasing or decreasing their transcription.
Has its own promoter and is transcribed into a short mRNA, which is translated into a small protein.
Regulator protein binds to a region of the operon
Regulatory element
DNA sequence that impacts expression of gene that is adjacent [cis-acting]
beta-galactosidase gene
encodes a protein that normally breaks down, lactose, a common sugar in milk, into two pieces, glucose and galactose
Glucose= usable sugar form
ALSO called lactase
lactose operon
In the presence of lactose, concentrations of the enzymes responsible for lactose metabolism increase rapidly from a few molecules to thousands per cell.
The enzymes responsible for lactose metabolism are inducible, and lactose is the inducer.
lac Z, lac Y, lac A
In the lac operon, genes will be transcribed into the mRNA and eventually produce the enzymes required to break down the lactose
3 structural genes of lac operon
lac Z, lac Y, lac A
lacZ gene encodes β-galactosidase.
****Converts lactose to glucose and galactose
- lacY gene encodes permease.
*****Facilitates entry of lactose into bacterial cells
-lacA gene encodes transacetylase.
****Removes toxic by-products of lactose digestion
Lactose
glucose + galactose
Galactose
a monosaccharide; part of the disaccharide lactose
Glucose
components of lac operon
Regulatory region
Promoter
Operator: DNA sequence
**REMEMBER repressor binds to operator*******
Structural genes:
1. Beta Galactosidase gene [lac Z]---metabolizes lactose
2. Permease gene [lac Y]--makes membrane permeable to allow for influx of lactose
3. Transacetylase gene [lac A]---breaks down toxic byproducts
How is lactose expressed?
expressed at a basal level
-There is enough lactose to get into cell
-If operon highly expressed--allow greater influx of lactose.
**Lactose binds to lactose binding site.
no lactose present in lac operon
lacZYA off
Repressor CANNOT bind to promoter.
RNA polymerase [think of it like a train and the structural genes are the track] is blocked so CANNOT synthesize RNA of structural genes;
SYSTEM IS OFF; prevents transcription
Allosteric reaction: repressor alters shape and DOES NOT bind to operator. Sigma factor recognizes promoter BUT RNA polymerase cannot get through.
SO RNA POL recognizes promoter and synthesizes RNA from structural genes
Lactose present
lactose binds to repressor and inactivates the repressor protein so that transcription can occur
SYSTEM IS ON so that is why lactose induces expression of operon
NO binding to repressor
Allosteric reaction;
mutant repressor gene when Lactose absent
Normally, regulatory repressor that represses expression of lac Z gene.
Lac I encodes the repressor--> Decreases expression of Beta galactosidase.
Mutant repressor Gene When Lactose present
Genetic Dissection experiments
Created mutations and looked to see what happens.
Mutation to lac I gene [repressor]; Loses repressor function--> Will have EXPRESSION but NO repression--> Operon ALWAYS ON SO System is always ON.
Lac O
operator which is the binding site for a protein [lac repressor]
MUTATION of operator--> RNA POL will not bind to operator REGARDLESS if lactose present or not.
DNA binding protein normally recognizes DNA sequences [operator] BUT sense operator is mutated--> DNA binding protein does NOT recognize mutated sequence
Oc= constitutive operator; ALWAYS on.
Genetic Proof of Operon Model
Monod wanted to determine cis and trans-acting
SO he created partially diploid [merozygote] bacteria for genetic dissection experiments
Add genes to F factor [plasmid or extra chromosomal DNA]
Determined B-galactidose activity in presence or absence of lactose
Partial diploid [merozygote]
a strain of bacteria containing F' factor genes
too much lactose?
Catabolite activating protein [CAP] exerts positive control over lac operon
So if glucose is increased--> cAMP is decreased [TOO much glucose means that CAP has NO cAMP to bind to SO if you have too much, you do not want the operon to keep going, so you turn it off]
No glucose?--> Increased cAMP
**Operon will be ON because you want to bring in more lactose to get glucose
catabolite activator protein [CAP]
In order to bind to the promoter, CAP must be bound to cyclic adenosine monophosphate [cAMP]. [cAMP allows CAP to bind efficiently]
Binding of the CAP-cAMP complex to the promoter region increases the efficiency of binding of RNA polymerase to the promoter
****The level of cAMP is dependent on the enzyme adenyl cyclase.
Glucose inhibits the activity of adenyl cyclase, which catalyzes the conversion of ATP to cAMP and thus prevents CAP from binding when glucose is present
The Tryptophan [trp] Operon in E. coli Is a Repressible Gene System
Tryptophan production is REPRESSIBLE [on all the time
and must be turned off by repressor SO "On until turned off."]
-
Five contiguous genes on E. coli chromosome encode
enzymes for tryptophan synthesis.
-
When tryptophan [corepressor] is present:
-Repressor and tryptophan complex attain new conformation.
-
Binds to operator, repressing transcription—enzymes not made
trp= corepressor so binds to repressor and turns system off; Allosteric reaction occurs when corepressor bound to repressor AND can bind to operator and to RNA so system is turned off.
Eukaryotes
Tightly controlled to express required levels of gene products
Prokaryotic gene regulation occurs primarily at transcription initiation.
Eukaryotic gene regulation occurs at different levels.
Lack operons
CORE promoters must be in fixed positions
enhancers and silencers
speed up [enhancer] or slow down the rate of transcription [silencers]
Enhancers and silencers regulate transcription of eukaryotic genes.
Cis-acting transcription regulatory elements-NOT IN FIXED LOCATIONS!
Transcription factors
A regulatory protein that binds to DNA and affects transcription of specific genes.
Transcription regulatory proteins- Trans-acting Factors
Target cis-acting sites of genes regulating expression
Activators increase transcription initiation.
Repressors decrease transcription initiation.
Multiple transcription factors bind to several different enhancers and promoter elements and fine-tune the level of transcription initiation.
Transcription activators and repressors
bring changes to RNA Pol II transcription.
DNA looping delivers activators, repressors, and general transcription factors to promoter vicinity.
Recruitment model: Enhancers and silencer elements act as donors and affect regulatory proteins at gene promoters'
Activators: Proteins that bind enhancers; increase transcription
Repressors: Bind silences; decrease transcription levels.
coactivators
proteins that increase the rate of transcription but do not directly bind to the DNA itself
Interact with proteins and enable activators to make contact with promoter-bound factors
Coactivators form complex "enhanceosome"****
A transcription factor may become active only when modified structurally [e.g. phosphorylation].
enhanceosome
a large protein complex that acts synergistically to activate transcription
Binds to enhancer
TATA box is recognized by TF2D
Human Metallothionein IIA Gene [
hMTIIA]
Example of how a gene can be transcriptionally regulated due to interplay of:
•
1. Promoters
2. Enhancer elements
3. Transcription factors that bind to them
Product of hMTIIA
Protein binds to heavy metals and protects cells from toxic effects.
-
Protects cells from oxidative
stress
-
Expressed in low levels in all cells
-
Transcribed at high levels when exposed to heavy
metals---> ***Heavy metals will increase transcription of metallothionine
Ex: Glofish [Fish glow due to constitutive promoter so ALWAYS ON no matter what]
RNA interference [RNAi]
A technique to silence the expression of selected genes in nonmammalian organisms.
The method uses synthetic double-stranded RNA molecules matching the sequence of a particular gene to trigger the breakdown of the gene's messenger RNA.
Small interfering RNA---> Viral or genetically engineered dsRNA is an exact match to gene of interest; develop and inject into cytoplasm
Dicer protein cuts into tiny pieces of dsRNA and separates the strands--> mRNA gets degraded and prevents translation
micro RNA
Naturally occurring
Gets into nucleus
Single stranded RNA that forms secondary structure [End up with 20 base pairs of dsRNA]
BIND to RISC; Exact match--> degrades mRNA mismatch--> miRNA stays bound to mRNA and INHIBITS translation
Notes from Chapter 12 Reading
...
Gene expression controlled at different levels
Includes:
Alteration of gene structure
Transcription
mRNA processing
MRNA stability
translation
post-translational modification
Much of gene regulation takes place through the action of regulatory gene products that recognize and bind to regulatory elements
Genes in bacterial cells
Typically clustered into operons--groups of functionally related structural genes and the sequences that control their transcription.
Structural genes in an operon are transcribed together as a single mRNA molecule
negative control of transcription
occurs when a regulatory protein called a repressor binds to DNA and shuts down transcription
positive control of transcription
a regulatory mechanism that starts transcription through an activator protein that activates the binding of RNA polymerase to DNA
Inducible operons
transcription is usually off [inhibited] and needs to be turned on [induced]
The regulator protein is a repressor that binds to the operator and prevents transcription of structural genes
when the inducer is present, it BINDS to the regulator, thereby making the regulator unable to bind to the operator. Transcription takes place
Repressible operons
transcription is normally on and needs to be turned off
1. Regulator protein is an inactive repressor, unable to bind to the operator
2. Transcription of the structural genes therefore takes place
3. Levels of product build up
4. Product binds to regulator protein...
5. ...Making it active and able to bind to the operator
6. AND THUS prevents transcription
Lac operon of E.coli
negative inducible operon.
In absence of lactose, repressor binds to the operator and prevents the transcription of genes that encode B-galactosidase, permease, and transacetylase
in presence of lactose, some of it converts to allolactose [inducer], which binds to the repressor and makes it inactive, allowing the structural genes to be transcribed
Positive control in the lac operon
regulated by catabolite activator protein [CAP]
When complexed with cAMP, the catabolite activator protein binds to a site in or near the promoter and stimulates the transcription of the structural genes
levels of cAMP are inversely correlated with glucose, SO LOW levels of glucose stimulate transcription and HIGH levels inhibit transcription****
trp operon [E. coli]
negative repressible operon that controls the biosynthesis of tryptophan
in repressible operon, transcription normally turned ON and must be repressed, this is accomplished through the binding of tryptophan to the repressor, which renders the repressor active.
The active repressor binds to the operator and prevents RNA polymerase from transcribing structural genes
Eukaryotic vs prokaryotic gene regulation
Eukaryotes= absence of operons, Presence of chromatins, and presence of nuclear membrane
Eukaryotic chromatin structure
Chromatin structure is directly related to the control of gene expression [represses it]
Chromatin structure can be altered by chromatin remodeling complexes that reposition nucleosomes and by modifications of histone protein INCLUDING:
Acetylation
Phosphorylation
Methylation
Methylation of DNA affects transcription
Initiation of Eukaryotic Transcription
Controlled by general transcription factors that assemble into the basal transcription apparatus and by transcriptional regulator proteins that stimulate or repress normal levels of transcription by binding to regulatory promoters and enhancers.
Enhancers function
affect transcription of distant genes
Regulatory proteins bind to enhancers and interact with the basal transcription apparatus by causing the intervening DNA to loop out
Insulators function
limit the action of enhancers by blocking their action in a position dependent manner
Coordinately Controlled Genes in Eukaryotes
Respond to the same factors because they have common response elements that are stimulated by the same transcriptional activator
Gene expression in eukaryotic cells
Can be influenced by RNA processing and by changes in mRNA stability
5' cap, the poly [A] tail, the 5' UTR, the coding region, and sequences in the 3' UTR are important in controlling the stability of eukaryotic mRNA
RNA interference
Plays important role in eukaryotic gene regulation. Initiated by double stranded RNA molecules that are cleaved and processed.
Small RNA molecules [siRNAs and miRNAs] combine with proteins and bind to sequences on mRNA or DNA
These complexes cleave RNA, inhibit translation, affect RNA degradation, and silence transcription
posttranslational modification
may play a role in the regulation of gene expression
Arabidopsis thaliana [A. thaliana]
possess a number of characteristics that make it an ideal model genetic organism
Epigenetic effects on gene expression
inherited changes in gene expression NOT DUE to changes in the DNA base sequence--are frequently causes by DNA methylation and alterations in chromatin structure
Epigenetic changes are STABLE but can be affected by environmental factors
Many epigenetic phenotypes result from changes to chromatin structure
Epigenetics effects occur through DNA methylation, histone modifications, and RNA molecules
Paramutation
A heritable alteration of one allele by another allele without any change in the DNA sequence.
Early life experiences can produce epigenetic changes
can have long lasting effects on behavior
Environmental chemicals may produce epigenetic effects that are passed to later generations
Phenotypic differences between genetically identical monozygotic twins may result from epigenetic effects
Epigenome
complete set of chromatin modifications possessed by an individual organism
Operon
single transcriptional unit that includes a series of structural genes, a promoter, and an operator.
Comparison of gene expression in bacteria and eukaryotes
Not all genes are expressed at ALL times in ALL situations
Can have same genes throughout body but different genes expressed through different parts of the body.
MANY prokaryotic gene products are present continuously at LOW levels; these can increase as needed.
In multicellular eukaryotes, differential gene expression is also essential and is at the heart of embryonic development and maintenance of the adult state.
Tricks for Gene activity problems [Presence/absence of lactose]
Z-: NEVER make functional B-galactosidase SO B-gal activity with or without lactose present
I-: Mutant repressor, ALWAYS make B-galactosidase
Oc= constitutive operator; Repressor cannot bind so never turned off; ALWAYS produces nonfunctional B-gal.
Ic: super-repressor; CANNOT be stopped from binding to operator EXCEPT constitutive operator can stop super repressor.---> ALWAYS binds to operator so NO product
Lac I vs. Lac O
Lac I works in trans
Lac O works in cis
Difference between cis vs trans-acting elements
By using different combinations of mutations on the bacterial and plasmid DNA, Jacob and Monod determined that some parts of the lac operon are cis acting [able to control the expression of genes only when on the same piece of DNA], whereas other parts are trans acting [able to control the expression of genes on other DNA molecules].
Understanding cis and trans-acting element
For E. coli strains with the lac genotypes given below, use a plus sign [+] to indicate the synthesis of β-galactosidase and permease and a minus sign [−] to indicate no synthesis of the enzymes.