Gene Regulation Flashcards
Examples of Transcriptional gene regulation
- Lac Operon
- Trp Operon
Gene Expression
The process that heritable information in a gene is made into a functional gene product (Protein or RNA)
Gene Regulation
The process of controlling gene expression
Where can the mechanisms of gene regulation occur
Can occur during any part of gene expression
Includes:
1. Transcription
2. RNA processes
3. Translation
4. Post translational modifications
Euk RNAs + regulation
Eukaryotic RNAs have introns that need to be spliced –> controlling gene expression can occur during processing of RNA
Translation Regulation
Sometimes proteins are non-functional when first translated – need to be modified to function
Gene regulation in bacteria
In bacteria – regulating gene expression is mainly done at the transcriptional level
***Bacteria – Transcription + translation are couples = most of bacteria gene regulation is at the transcriptional level
Lactose Metabolism
Background: Preferred energy source in bacteria = glucose
IF no glucose THEAN bacteria can use other energy sources such as Lactose
Lactose –> Galactose + glucose
***It is more effective to use glucose if have glucose –> if you have glucose then won’t make the enzymes required to turn on lactose pathway when lactose is present
Prefered energy source in bacteria
Glucose
Genes on Lack operon
LacI
CAP binding site
Promoter
Oportaor
LacZ
LacY
LacA
Lac Z, Y, A = Invloved in lactose metabolism –> Transcribed together same mRNA –> OPERON
- Each (LacZ, LacY, LacA) have own ribosome binding site
Constitutive Gene expression
Expressed all of the time at constant levels
Lac Z
Codes for B-galactoside – enzyme that converts lactose to alolactose + galactose + glucose
LacY
Codes for Lactose permease – protein that allows lactose to enter the cell
Lac A
Do not know what it codes for – knock out doesn’t seem to have effect
Opator in Lac Operon
Between Promoter + Lac Z –> have operator domain
Function: Where repressor can bind to
CAP binding site
In Lac operon – upstream of promoter
Function: Place where CAP can bind
Operon
Multiple coding regions on same mRNA
Expression of LacI and CAP
LacI + CAP = expressed at consistent levels throughout cell = constitutive gene expression
Promoter in Lac operon
Upstream of LacZ gene
- When RNA polymerase binds to promoter – transcribed and get 3 genes
+1 start site is just downstream of this
Regulatory genes in Lac operon
- Lac I – ecodes lac repressor
- Upsteam of Lac operon (not officially part of lac operon – has its own promoter + creates own transcript)
- CAP – Catabolic activator protein
- Found elsewhere on the circular chromosome – has own promoter
Lac operon repressor (Overall)
No lactose –> Lac repressor binds = no transcription
Yes Lactose –> Allolactase binds to repressor = induce transcription
Lac operon repressor
Repressor = can bind to operator sequence
WHEN binds it gets in the way of RNA polymerase that is trying to sit on the promoter and induce gene expression
When repressor is bound to operator = transcription is blocked
WHEN lactose is present –> Some amount of the lactose is converted to allolactose –> Allolactose can bind to the repressor = prevents the repressor from binding to the Operator site = NOW RNA polymerase can sit on the promoter and begin transcription
Promoter + Operator sequnce of Lac operon
Promoter has:
-35 abd -10 sequence
+1 start site = beginning of the operator sequence
Transcription of Lac Operon
RNA polymerase = begins to transcribe the Lac operon BUT repressor bound to the operator sequence prevents +1 from being accesible to RNA polymerase = blocks transcription
Type of regultion in Lac Operon
Lac operon = example of negative regulator
***Uses negative regulation to control gene expression
Negative Regulation
When binding of regulatory protein blocks transcription
Allolactse is an
Inducer – A small molecule that activates gene expression
Induce
A small molecule that activates gene expression
CAP in Lac Operon
CAP/cAMP complex – when bound CAP binding site = RNA polymerase is recruited to the promoter sequence = get HIGH transcription
- Promotes transcription
CAP activation
When CAP is transcribed into a protein CAP is inactivated in the current form BUT when bound to cAMP = CAP can bind to the CAP binding site
cAMP
A signaling molecule produced by glucose metabolism
Glucose level vs. cAMP level
Glucose is high = cAMP is low –> no cAMP = no bind to CAP binding site
Glucose is low = cAMP is high –> can bind to CAP binding site = get transcription
CAP Binding to CAP binding site
CAP binding to CAP binding site = causes DNA to loop around = making -10 and -35 more accesible to RNA polymerase –> RNA polymerase is recruited to the promoter = get transcription
Image of Lac operon
What type of regulator is CAP
CAP is an example of a positive regulator
Positive regulation
When binding of a regulatory protein activates transcription
cAMP is an…
Inducer – cAMP allows CAP to bind to the binding site
Overall Lac operon
- Lactose + No glucose = Highest transcription
- Lactose = have allolactase –> Allolactase binds to Repressor = prevents the repressor from binding to operator = get transcription
- No glucose = cAMP increases = CAP binds to cAMP = CAP binds to the binding site = recruits RNA polymerase = transcription occurs
- Lactose and Glucose = some transription – Even though CAP is not helping bring RNA polymerase to promoter RNA polymerase can still find promoter and and produce some transcript
- Glucose is high = low cAMP = CAP won’t bind to recruit RNA polymerase
- Lactose = have some allolactose to bind to repressor
- NO Lactose + Glucose = No transcription
- No Lactose – repressor binds to operator
- High glucose = low cAMP = CAP won’t bind to site = not recruiting RNA polymerase
- No RNA polymerase + repressor blocking transcription = No transcription
- No Lactose + No glucose = No transcription
- No glucose = high cAMP = CAP binds to CAP binding site
BUT - NO lActose = reprssor is bound to operator = even though RNA polymerase is being recruited the reprssor is blocking transcription –> repressor is good at blocking transcription = when reprssor is bound even when have CAP = transcription is blocked
- No glucose = high cAMP = CAP binds to CAP binding site
Exception in No Lactose + No glucose
Have leaky expression – will produce a few transcripts
- Transcripts can sneak off = small amount of product = leaky expression
Putting Lac Operon together
Start with No lactose BUT reprssor binds to operator sequence because of leaky gene expresses small amount of B-galactosidase + Lactose permase will be produced
Lactose present can enter the cell through lactose permease – in cell B=Galasidase can convert it to allolactase
Allolactase binds to reressor = falls off operator –> promotes transcription of operon = produce more B-galactosidase + permase
Transcription of operon continues and make permase + B-galactosidease as long as lactose is present in the media
When cell runs out of Lactose = no allolactor = repressor binds to operator and prevents transcription
Over time proteins will be degraded –> go back to small amount of permase + galactosidase because of leaky expression
What is the Trp operon an example of
Example of gene regulation through attenuation
Tryptophan
Essential Amino Acid
***Sleep inducing compound after eating Turkey
Producing Tryptophan
Mammals can’t produce own Trp – need to be obtained through foods BUT bacteria can synthesize Trp
E.coli – the enzymes used for Trp Synthesis = found in Trp operon
Bacteria – can also use Trp that they find in the environment –> they don’t always need to produce enzymes involved in Trp synthesis = Trp operon is regulated
Regulation of Trp Operon
Expression of the Trp Operon is regulated in 2 ways:
1. Negative regulation (using a Co-repressor)
2. Attenuation
Trp Operon (gene)
Trp repressor – Translates into protein Trp Repressor
- Represor gene (not on Trp operon)
Promoter – recruites RNA polymerase to produce RNA
Operator – Where Trp reprssor binds
Trp Leader peptide – Transcribed to mRNA (Part of annetuation process)
5 genes that encode enzymes needed for Trp Biosynetshsis
5 genes that encode enzymes needed for Trp Biosynetshsis
Co transcribed on the same RNA = p[eron of Trp Biosynthesis genes
Once they are translated to proteins – the proteins are part of complex required to convert Chorsimate to Trp
Trp repressor regulation
Trp Repressor can’t bind to operator sequence – it id in inactuve form when it is first translated into protein
THEN Trp can bind to Trp repressor – when binds = activates represor = allows represor to bind to operator sequence
When bound RNA polymerase might be recruited to the promoter but it can’t push represor = No transcription of downstream genes
Overall: Lowers transcription – lowers amount of transcript by 70%
Trp Repressor form of regulatino
Negative regulation – Bound regulatory protein prevents transcription of downstream genes
***one of the two steps involved in regulating Trp biosynthesis
Trp is a …
Co- repressor – because Trp and Trp repressor binds to operator
Trp Repressor regulation (Overall)
Trp is low = Transcription is High
Trp us high = Transcription is low
Transcription + Translation in Bacteria
Transcription + Translation are coupled – RNA polymerase produces a transcript that is being co-translated before transcripts are done being made
***Important for second regulatory step of attenutation in Trp operon
Attenuation Definition
To lower affect of something – Lowers expression of genes
What does Attenuation use in Trp Operon
Attenuation = accomplished through Trp Leader peptide (TLP)
TLP structure
RNA produces has TLP – has 4 regions of TLP THEN stop codon
TLP = only 14 Amino Acids long
Region in TLP
Regions have complementary sequences to each other –> Complementary sequences can form stem loop structures in the mRNA
MEANS – there are 2 possible secondary structures that are formed in the mRNAs
Secondary structures formed in mRNA
2 possibilities:
1. Attenuation form –> When Stem loop structure is formed between regions 3 and 4 AND between 1 and 2
- Anti-Termination –> Stem loop between regions 2 and 3
Region 1 in TLP
Region 1 contains 2 trp codons (Need tRNA with Trp to produce TLP)
Attentuation When Trp levels are high
Ribsome binds to initate protein synthesis of RNA emerging from RNA polymerase – because have Trp in cells = have charged tRNA with trp ready for protein synthesis
Ribosome can produce the peptide of TLP including the trp residues encoded by Trp codons in region 1
As ribsome goes down mRNA it covers 1 and 2 region of the LPS
Polymerase is ahead – transcaribes regions 3 + 4 –> regions 3 + 4 can form Stem Loop while the ribosome is completing translation
Stem loop of 3 + 4 = followed by a series of U resides
Stem loop + poly U sequences (AT rich region) = forms transcription termination sequence = allows RNA polymerase to fall off transcript = prevents transcription of things down stream
***Rho independent form of transcription termination
Attentuation with high trp (Overall)
High Trp = have charged tRNA high = Ribosome produces protein to make LPS = Stem loop forms between 3 + 4 = Stop Trp synthesis
- High Trp = Terminated = not produces Trp Biosynthesis enzymes
- High Trp = attentuation structure is encouraged in RNA = causes termination of transcript
Attenuation - Low Trp levels
Ribosome will binds to the newly synthesized mRNA and begin to produce the LPS BUT when reaches Trp codons in region 1 –> ribsome requires charged tRNA with Trp residue
BUT since in low Trp = most tRNA won’t have Trp AA avalable to put into LPS –> Ribosome is stalled siting on region 1 waiting for charged tRNA
RNA polymerase is transcribing and continues to transcrived regions 2 + 3 + 4 –> regions 2 + 3 can form stem loop structure – forms anti-attentiation structure
When Anti-attentuation structure is formed the terminater signal made by 3 + 4 = can’t signal the end of transcription = RNA polymerase will continue transcribing genes TRP e,d,c,a
Low Trp Levels Attentuation overall
Promote stalling of ribosome = promotes formation for anti-attenutaion strucure = allow transcription to proceed
Regulation of Trp Operon (overall)
Trp is low –> Transcription proceeds – enzyme synthesis occurs allowing Trp biosynethsis
- Trp repressor = inactivated = allows transcription to proceed
- Anti antenuation structire on mRNA = allow transcription to proceed
Trp is high
- Activates trp repressor = allowing it to bind to operator = prevents transcription
- Have attenuation structure on mRNA –> Produces Stem loop termination sequqnece early = transcription is halted = no enzyme syntehsized = cells won’t produce Trp
How much does Trp repressor prevent Transcriotion
Prevents 70% of transcription = need additional regulation to reduce transcription level
Additional regulation - accomplished by promoting attenuation structure
What is Trp an example of
The Trp operon is an example of how attentuation regulates gene expression by controlling transcription
Use of Attenuation
Transcriotional regulation through attenuation is widely used in Prokaryotes + Archea
- Level of transcriptional regultions is used in prokaryotes + Archea = suggests it is evolutionary ancient mechanism of gene regulation
Second use of attenuation
Attenuation might also be used to regulate protein translation –> IF mRNA makes attenuation structure that involoves the Ribosome binding site –> could prevent the ribsome from binding to mRNA = block translation
***Active area of reserach – looking for genes regulation by attenuation to conrtol translation
Attenuation (Euk)
Attenuation might be important mechanism of gene regulation in Euk where transcriotion + Translation are not couples
Gene regulation Eukaryotes
Mechansim leveles of gene regulation in Eukaryotes is much less well understood
Themes in biology
Themes in biology tend to repeat themselves –> things learned in lac operon can be inform us about how more complex beings regulate gene expression
What does the LacOperon and The Trp Operon show is
The Lac Operon and the Trp Operon show us examples of what can be used to regulate gene expression
Why doe organisms need to regulate gene expression
- Cost energy to make proteins
- If expressed all proteins then the cell would be overloaded (no space)
- Different organs and tissues need different thinks
- Need different things in different environments
Genes that are not regulated
Not all genes are regulated – some are on all of the time (Constitutive genes)
There are things that we always need
Example - Ubiquitin + ATPase + RNA polymerase + Oxidative phosphorylation + Histones + Ribosomes + DNA repair paths + Cell cycle enzymes
Ways to regulate gene activity
- Transcription - block transcription (Block or alter transcription rate)
- Translation
- Block or change translation rate
- Change mRNA life span (stability) – could degrade RNA (block translation by getting rid of mRNA)
- Post-tranlation – modify proteins to activate or inactivate (chemical modifications)
If you were a bacteria - what would be the most efficonet way to turn off a specific gene
Block Transcription – save energy (not making transcript OR protein)
ALSO it is hard to block all transcripts from being translated (one can be translated and make lots of proteins)
1 DNA makes
1 DNA makes many transcripts
1 Transcripts makes many proteins
Glucose
Easy energy source – goes through glycolysis to make pyruvate
***ALL other energy sources require more ATP input and or fewer ATP output
Lactose
Alternate energy source
Overall:
Brought in by Lactose Permase THEN Lac Z that makes B-galactosidase
B-galactosidase = coverts lactose –> Glucose + galactise
- Side product = Makes allolactase – can be converted to glucose
B-galactosidase
Lactose –> glucose + galactose
AND
Lactose –> Allolactase
Lac operon (overall)
Lactose and no glucose –> ++++ – Tons of expression
Lactose and glucose –> ++ – Little expression
- Prefer glucose but uses some lactose
No Lactose and no glucose –> +- – no expression but have leaky expression
No Lactose and Glucose –> – no expression
Lactose effect on Lac operon
Lactose induces expression of the Lac operon
How does Lac Operon sense the presense of Lactose
Sense the presence of lactose through allolactose
Lac repressor binding to operator
Lac repressor binds to operator sequence with high affinity (binds tightly)
***When bound RNA polymerase can’t find the transcription start site – covers start site
- RNA polymerase goes to the -10 and -35 elements but can’t do anything
Positive vs. Negitive regulation
Positive – Protein binds and promotes transcription
Negative – protein binds and prevents transcription
Answer: Negitive regulator
Answer: Not bound to the operator
Use of cAMP
Activates genes when low glucose
How does CAP recruit RNA polymerase
CAP bound to the CAP binding sequence –> taunts RNA polymerase by exposing -10 and -35 regions
***Promotes expression by exposing promoter to RNA polymerase
Answer: Positive Regulation
Lac Operon Outcomes (Summary)
Lactose + No glucose – Highest level of transcription
- Low glucose = High cAMP = CAP bound –> Transcription
- Lactose –> Have Allolactose –> Repressor = not bound –> Transcripotion
Lactose + Glucose – Some transcription BUT lower
- High lactose –> Repressor not bound –> Transcription
- High glucose = low cAMP = CAP Not bound –> No transcription
No Lactose + No glucose –> No transcription BUT leaky expression
- No lactose = repressor bound –> No transcription
- Low glucose = High cAMP = RNA polymerase is recruited byt repessor is good at blocking BUT sometimes RNA polymerase goes befire repressor gets there = get transcription before reprssor –> make some protein = get some expression
No lactose + glucose – No Transcrioption
- High glucose = CAP not bound –> No transcription
- No lactose = No allolactose = repressor bound –> no Transcription
Leaky Expression
There is some basal very low level of expression
How does Leaky expression work
No Lactose + No glucose –> No transcription BUT leaky expression
- No lactose = repressor bound –> No transcription
- Low glucose = High cAMP = RNA polymerase is recruited byt repessor is good at blocking BUT sometimes RNA polymerase goes befire repressor gets there = get transcription before reprssor –> make some protein = get some expression
***One transcript can be translated multiple times allow a small amount of protein expression
Lac Operon cycle
Start = no Lactose + get some B-galactosidase
THEN – add lactose –> Lactose enters the cell through Permease AND the lactose gets converted to allolactose by B-galactosidase
THEN Repressor not bound = get more expression = more allolactase = more repssors fall off = increase operon expression
THEN use all lactose = no more allolactose = repressor binds = block transcription
THEN Proteins are degraded expect some leaky expression = have some amount of proteins
Why might it be benefical to have leaky expression
If off all of the way and want to turn on in presence of lactose need some B-galactosidase to start the cycle
Overall: Hard to trun something off entriley
Answer: Constitutive
Repressor can’t bind = will get transcription all of the time
Answer: Repressed – Can’t make allolactose = repressor bound = always repressed
Use of Lac Operon
Lac Operon = provides lots for biotechnology + medicine
Biotech use of Lac operon
Can engineer bacteria o express insiulin under the lac promoter/operator
- IPTG = mimics allolacase causing LacI to fall off the operator
- Engineer plasmid to have a prmoter + operator –> Add LacI repressor = get no insulin
- Making insulin costs energy = want no insulin to be made when the cells are young THEN add IPTG to knock repressor off THEN cells will start to make insulin
- Use Lac operon to turn on/off
***Can be adapted to any gene
Monitoring gene expression in Lab
Can use Lac Z – can see when something is turned on (when gene is on it turns blue)
Cells can be engineered to express B-galatosidase under any promoter of interest
THEN add X-galactose and you can visualize the cells that are expressing LacZ (and infer about the regulation of the gene of interest)
Affintioty of Trp Repressor
Have low transcriptions BUT repressor has low affinity for operator = can be knocked off = can get some transcription but at a low rate
Probability of Trp codons next to each other
2 Trp next to each other is rare – used for regulation
Where is attenutaion used
Not limited to bacteria
Gene regulation Tool Kit
Activator – A regulatory protein that promotes gene expression
Repressor – A regulatory protein that prevents gene expression
Inducer – A small molecule that promotes gene expression
Co-repressor - a small molecule that prevents gene expression (typically works with a repressor)
Signalling molecule – a small molecule that transmits signals to regulatory systems (cell communication)
Regulatory Sequence – Sequences on DNA/RNA that control gene expression (Ex. promoters + operators + CAP binding site)
High Trp = turn off operon because induce termination sequence in RNA
***In living cells don’t get stem loop between 1 and 2 because ribosome is covering them
Transcription Termination (pro)
Transcription ends when a Stem Loop structure forms in the RNA followed by AT rich sequence – Need AT rich region after
***If just Stem loop then RNA polymerase won’t fall off
High vs. High Trp
Trp Operon sequence
TRP –> region 2 –> Region 3 –> Region 4 –> AT rich (lots of U)
Trp repressor – Will bind sometimes – some Trp = when find bind but not a ton = sometimes won’t bind –> somtimes blocked
Trl RNA structure formed –>
- Sometimes have tRNA = get attenuation structure
- Sometimes ribsome waits = SL 2 + 3 = Get anti attenutaion structure
Overall Expression – moderate
Lactose vs. trp expression
Lac operon = don’t want to express if have no lactose = no expression
Trp = you always have moderate expression
Use of Attenuation
Allows fine tuning of trp in cell
Better than waiting for an all or nothing approach to gene regulation
Answer: Get Anti-attenuation structire –> Promote transcription (ribosome stalls)
***System is no longer regulation by Trp – plays on
Positive vs. Negitive feedback loop
Positive = Product promotes expression to make more product
Negative = Product prevents more product from being made – product represses
Loop in Trp synthesis
Negative feedback loop – If increase Trp then repressor binds AND get attenuation structure
Loop in Lac Operon
Overall: Positive feedback
Increase lactose = increase allolactase = no repressor = get transcription THEN –> Increase Lac Z make allocatase = activate transcription more
Increase all = increase transcription = positive feedback loop because when produce Lac Z = produce more of what you need to get transcription
Moleculare in Trp Operon
Repressor –> trp Rpressor
Co-repressor –> Tro
Regulatory sequences –> Operator + Promoter +
***Trp Leader peptide = Don;t know – can argue part of regulatory sequence
