Final Exam Prep Flashcards
Transcription Initiation (Level of prokaryotic Gene Control)
Control if and how much housekeeping genes will be transcribed or not
- includes regulators
Types of Transcriptional Factors for Prokaryotes (Regulators)
Activators: increase expression (turn on or up genes) and increase the amount of transcription happening in these genes- These bind to
activator binding sites
Repressors: decrease expression (turn off or down genes);
decrease the amount of transcription happening in those genes and
binds to repressor binding sites
Positive Control
–> The gene is always off or little to no activity in the gene - Unless activator is there to help RNA polymerase bind to the promoter region
–> RNA polymerase has less affinity for the activator binding site, which is why the activator binds to RNA polymerase thus initiating transcription more often
–> Trans-acting factor must bind to cis-acting site in order for RNA polymerase to initiate transcription at the promoter
Negative Control
–> Gene is always on unless repressor is there to prevent RNA polymerase from binding to the promoter
–> Trans-acting repressor binds to cis-acting operator site to turn off transcription
Induction
–> Interaction of inducer molecule with the gene’s regulatory protein to enhance/turn on transcription
–> Negative control = Inducer changes the shape of the repressor so that it is no longer able to block RNA polymerase thus it can resume transcription
–> Positive control = The presence of the inducer turns an inactive activator to be active so that it can bind to RNA polymerase to help start transcription
Repression
–> Negative control = another molecule turns the inactive repressor to become an active repressor thus the gene is turned OFF
–> Positive control = another molecule turns an inactive activator to become activated and thus the gene is turned ON
Bacteria vs Eukaryote Regulator sites
- Eukaryotes do not contain many repressors but rather activators
- Bacteria have activator and repressor binding sites
- If the sites are close to the gene = easier interaction
- if there are far from the gene = DNA loops so regulators can interact with RNA polymerase
(common in eukaryotes and called enhancers - DNA bends so that activation can still occur)
Architectural Regulators
- Regions that bend between the activator and promoter sites which create contact between them and initiate transcription
- Help with DNA looping and common in eukaryotes
Why do Regulators use co-activators or co-repressors?
- coactivator helps bind the activator and RNA polymerase so that they can directly interact
- corepressor blocks activator from interacting with the RNA polymerase and prevents transcription
Both are very specific for particular genes
Insulators
Promoters are adjacently situated on a gene as transcriptions would occur upstream or downstream and so if genes are close together, insulators may be between them to prevent regulators from acting on the wrong promoter regions
Combinatorial Control of Regulators
- Use of specific combinations of regulatory proteins to control gene expression
- some regulators are shared for different regulatory binding sites since some genes need to be turned on at the same time
- Some are unique to the binding site because you might need these genes less than others
- Combinations help to turn on genes fully
Post-transcriptional process (level of gene expression control)
Includes:
- mRNA splicing: preRNA needs to have sections spliced before it can be used for translation
-modification of RNA: capping and poly A tail and RNA editing
(e.g., attenuation which is mRNA stopped before it is finished being transcribed)
Post transcriptional Stability (Level of gene expression control)
- If preRNA is made but not needed yet, then it can be silenced
-miRNAs are made temporarily and form duplexes with the target RNA - used in developmental timing, repressing transposons and destroying invading RNA viruses
-If preRNA is made but not needed yet, then interference to remove
-dicer cuts miRNAs into siRNAs which can be used to bind to mRNA to stop it from being used so that the function of the gene can be studied without making a mutant organism
-process called RNAi (RNA interference)
-DNA eventually destroyed
How do Regulator Molecules Interact with DNA in Prokaryotes?
- Through structural motifs (sequences)
- regulatory proteins often appear as dimers with short, inverted repeats as their structural motif
Helix-turn-helix motif
- often used by bacterial regulators
-the recognition helix inserts into the major groove of DNA
-INTRINSICALLY UNSTABLE AND PART OF A LARGER MOLECULE - Seen in lactose repressor
Homeodomain motif
-conserved 60 amino acid sequence motif
-repeats in many proteins
- 3 alpha helices that interact with minor groove
-dimers come in from both sides of DNA and hold it together
Leucine zipper motifs
● Interaction between 2 proteins
such as transcriptional factors -
leucine in every 7th position
● Part of dimer interacts w/ DNA
● Other part of dimer has leucine at
every 7th position
- Leucine interacts w/ other
leucines on the other part
of the protein dimer and
holds it together (leucine
acts as a zipper region)
- Allows DNA to open and
close flexibly
Viral Replication Strategies for Viruses
Lytic:
- Viruses that enter the cell, replicates, then leaves the cell
Lysogenic:
- Viruses that incorporate themselves into chromosomes of the cell, cell
division occurs, viral genetic material gets replicated and stays in the
cell, then it can choose to become lytic
Lytic Cycle
- Takes place by producing phage genomes and protein particles that are assembled into progeny phages (class 1 = 5 genes with RNA polymerase and host interference, class 2 = 7 genes with DNA synthesis and lysozyme and class 3 = 12 genes with heads and tails and DNA maturation)
-Phage lytic development
proceeds by a regulatory cascade where a gene product at each stage is needed for expression of the genes at the next stage
Stages of Lytic Cycle
Early: Phage genes are transcribed by host RNA polymerase (regulator gene products: RNA POLYMERASE, sigma factor or antitermination factor needed)
Middle: Early product causes transcription of middle genes (regulator gene products: sigma factor or antitermination factor; structural genes products: replication enzymes)
Late: Middle product causes transcription of late genes (structural gene products: phage components)
Lysogenic Phage
- Lysogeny is where they recombine their genetic material into the chromosome
-when they recombine they are called prophage (dormant/ static for an undetermined period of viral replication stages until an environmental stage that induces lytic cycle)
How can Viruses switch from one pathway to another?
- Low nutrient levels and high MOI → makes viruses stay in their lysogenic stage
- Prophage is incorporated into host cell chromosome
- Prophage leaves when host cell is under stress and goes back to the lytic cycle
Lambda
-A bacteriophage
- bacteriophage chromosome is circular when not integrated into host
- lambda Can integrate into E.coli chromosome at specific locations via recombination at att site- linear form
- E.coli has Gene for galactose and biotin → once lambda integrates there, its chromosome becomes linear
Pre (lambda)
- promoter used for repressor establishment in lambda
- Transcribes to the left
- Makes mRNA that eventually becomes a repressor
PR (lambda)
- right promoter
- Transcribes to the right, transcribing the other strand
PL (lambda)
- left promoter
- Transcribes to the left
PRM (lambda)
- promoter used for repressor maintenance
- Makes mRNA that produces repressor maintenance
- Maintains the level of repressor
Pint
- promoter used for integration of lambda
- If lambda decides to be lysogenic it needs this promoter to integrate its DNA into the host genome
OR (lambda)
- right operator (3 sequences that repressor molecule can bind to)
- ORI
- OR2
- OR3
OL (lambda)
left operator that can bind to
- OL1
- OL2
- OL3
CRO (lambda)
regulator protein, in high concentration when lambda is lytic
cI (lambda)
- repressor protein
- Represses the lytic cycle
- High concentration of cI → lambda will be lysogenic
cII
- transcriptional activator
- Helps w/ RNA pol binding
cIII
- binds to cII to help stabilize it
N- anti- terminator
- Take termination sites and turn them into non- termination sites
Int- integrase
- Excision and integrase protein are both needed to integrate lamda’s DNA
into the host genome
Lytic Cycle of Lambda
- Lamda infects bacterial cell → bacterial RNA pol recognize PR and Pl (the right and left promoter)→ transcription starts
- Cro, cII, N are made
- Promoters that have to be recognized by the host RNA pol - Cro binds to operator sites OR3 and OL3 first and helps RNA pol to bind
to promoter PR and PL and allows more transcription from PR and PL to take place
- PRM transcription is inhibited
● N is anti- terminator (at left and right) → terminator is a different
configuration → RNA pol can no longer recognize it → allowing RNA pol
to travel through the terminator and makes all the other gene necessary
for the lytic cycle including the head and tail protein for the viral particle
● cII helps RNA pol to recognize PRE, transcribes towards left
● Make cI the repressor - Cl binds to OR1 and OL1 → turns off all previous transcription bc it inhibits RNA pol from binding to PR and PL but helps RNA pol to bind to Prm and starts transcription from PRM
● Now auto- regulating its own transcription
How does the N protein work?
Binds to nut site and prevent RNA pol from recognizing the normal terminator bc it does
not form the correct shape → turns into lytic phage
● Once mRNA is transcribed through that region, it turns the next site into its
secondary structure (hairpin structure)
- Once made it can be recognized if the N gene is there
Lysogenic Cycle for Lambda
- When lamda infects the bacterial cell, the bacterial RNA pol recognizes Pr and PL and transcription starts
● Cro & cII and N are made - N is antiterminator so initial transcripts are elongated and helps stabilize cII
● cII is also made helps RNA pol recognize PRE
● This makes the cI the repressor
- Also bind to operator sites - cI binds to OR1 and OL which turns off all previous transcription bc its inhibits RNA pol from binding to PR and Pl but helps RNA pol to bind to PRM and starts transcription from PRM
● cI helps produce its transcript and now become auto-regulated
- Lambda will integrate bc cI will repress the lytic phase and keep it lysogenic
- Help it maintain and stay in the host chromosome
Operator sites for lambda regulators
- cI binds in different sequence to cro
- Repressor (cI) is a dimer and can bind to operator sequences → binds more strongly to OR1, than to OR2, then to OR3
- Binding is cooperative between OR1 and OR2
Switching network for Lysogeny Maintenance of lambda
● Cro and cl dictates whether the virus will be lytic or lysogenic
● Cro normally wins when the MOI (multiplicity of infection) is low or other conditions are not favourable
● cI wins when MOI is high
Lambda Integration into host’s chromosome
- Last step
-To be fully lysogenic the viral genetic material needs to integrate into the host’s chromosome - Integration happens by lambda integrates between the Biotin (bio) and Gal genes on the E. coli chromosome
- POP’(attP) and BOB’ (attB) sites are restored after excision
- IHF = integration host factor
How do Prophages turn Back into lytic phages?
- when cI gets cleaved or the stability of CII is compromised
- Things that cleave connector:
UV light (env’t) and Rec A (cell)
Why don’t Eukaryotes have 7 levels of control like prokaryotes?
- all genetic material is concentrated in the nucleus and there’s more of it for prokaryotes
- Eukaryotic DNA is tightly wound up by histone proteins
- Gene expression is controlled principally at the initiation of transcription
First Mechanism of Turning eukaryote genes on
After Y fork → When replication disrupts chromatin structure, after the Y fork has
passed, either chromatin can reform by histone binding or transcription factors can bind and prevent
chromatin formation
Second Mechanism of Turning eukaryote genes on
Transcription factors can bind to DNA outside of histones and recruit histone modifier
to open up gene region
-Either uni/ bi directionally until it reaches an insulator (boundary
element)
TAF
Transcription activating factors
TBF
TATA binding factor
TF
transcriptional factor
- activated by a cell or env’t signal and recruits the CBP-PCAF-HAT complex which binds to the SWI/SNF complex which is an ATP-dependent chromatin remodeling complex and brings RNA polymerase to region to start transcription
Eukaryote Activators
Molecule that determines the frequency of
transcription
● Usually upregulation
- Helps RNA pol increase
transcription
Chromatin remodeling
taking chromatin from closed complex → open complex
Eukaryote Repressors
- Protein that inhibits the expression of a
gene
● May act to prevent transcription by
binding to a regulator site in DNA or by preventing translation by binding to RNA
● Downregulation
- Decreases frequency of
transcription sites
-Eukaryotes do not have operator sites
but still have repressor sites that are
called regulatory sites
Positive control for eukaryotes
- Default state of genes that are under
positive control is that they cannot be
expressed unless a positive regulator
(usually an activator) is bound - Needs help so that genes can be transcribed
Classes of Activators in Eukaryotes
-True activators
-Antirepressors
-Architectural proteins
True Activators in Eukaryotes
● Function by making direct physical contact (protein → protein) w/ basal apparatus at promoter
● Either makes direct contact w/ RNA pol or one of the initiation factors at the
promoter site
Synthesis of protein (true activator)
- Activator can bind to RNA pol or enhancer sequence and turn on the gene
- If activator is not there, the second gene will not be able to function
Covalent modification of protein ( true activator)
- Activator is made but is inactive
- Covalent modification occurs to make it active (i.e phosphorylation)
Ligand binding (true activator)
- Two molecules come together to create the activator and activates it
- I.e hormone attaching to protein
Direct binding of inhibitors that sequester the protein or affect its ability to bind
to DNA (true activator)
- activator is made and binds to inhibitor (inhibited from functioning bc it is bound to inhibitor molecule
- Sequestered in the cytoplasm and does not affect nucleic activity
- The inhibitor needs to be able to be removed; then activator needs to be able to travel to the nucleus
Ability to select the correct binding for activation (true activator)
- Multiple proteins that can bind in different configurations but
only one configuration can initiate activation - Largely due to affinity
Cleavage from an inactive precursor (true activator) - true activator
- Repressor bound to active site
- Enzymatic activity cleaves the inactive precursor
Antirepressors - eukaryotes
● Function to recruit histone modification enzyme to bound activators to convert chromatin from closed to open state
Architectural proteins - eukaryotes
● Function to bend DNA
● Controls whether bound proteins can contact each other
● Do not directly bind to transcriptional/initiation mechanism
- Bind DNA so other proteins can create contact
- Help other activator proteins to initiate transcription
How Eukaryote True Activators Control Transcription
- During synthesis of protein
-By covalent modification of protein
-ligand binding
-Direct binding of inhibitors that sequester the protein or affect its ability to bind to DNA - Ability to select the correct binding for activation
- Cleavage from an inactive precursor
How Eukaryote Repressors Control Trancription
–> Sequestering an activator in cytoplasm (Bind to activator and prevent it from going where it needs to go - keeping it in cytoplasm rather than letting get to the nucleus)
–> Binding an activator and masking its activation domain (Binds to activator and prevents it from doing its function)
–> Being held in cytoplasm until needed (Repressor is stuck in the nucleus and Only when the activator is needed that it comes off the cytoplasm)
–> By competing w/ an activator for binding site
Mechanism of Activators and Repressors in Eukaryotes
- Some components of the transcriptional apparatus work by changing chromatin structure.
- Repression is achieved by affecting chromatin structure or by binding to and masking activators.
● Activators and repressors often have DNA- binding and activating functions in independent domains of the protein - not found in the same location
● Mediator complexes associate w/ RNA pol & replace activators/ co-activators
and basal factors
How Activators Interact with the Basal Apparatus in Eukaryotes
- a DNA-binding domain determines specificity for the target promoter or enhancer.
-The DNA-binding domain is responsible for localizing a transcription-activating domain in the proximity of the basal apparatus.
-An activator that works directly has a DNA-binding domain and an activating domain
-An activator that does not have an activating domain may work by binding a coactivator that has an activating domain.
-Several factors in the basal apparatus are targets with which activators or coactivators interact. - RNA polymerase may be associated with various alternative sets of transcription factors in the form of a holoenzyme complex.
-Mediator complexes associate with RNA polymerase and replace activators/co-activators and basal factors
Zinc finger Motif (Eukaryotes)
- A DNA-binding motif that typifies a class of transcription factor
Steroid receptor Motif (eukaryotes)
Transcription factors that are activated by binding of a steroid ligand
helix-turn-helix motif (eukaryotes)
The motif that describes an arrangement of two α-helices that form a site that binds to DNA, one fitting into the major groove of DNA and the other lying across it
homeodomain motif (eukaryotes)
-A DNA-binding motif that typifies a class of transcription factors.
helix-loop-helix motif (eukaryotes)
- The motif that is responsible for dimerization of a class of transcription factors called HLH proteins.
- A bHLH protein has a basic DNA-binding sequence close to the dimerization motif.
leucine zipper (eukaryotes)
– A dimerization motif that is found in a class of transcription factors.
bZIP (“basic zipper”) - eukaryotes
A bZIP protein has a basic DNA-binding region adjacent to a leucine zipper dimerization motif
chromatin remodelling (eukaryotes)
The energy-dependent displacement or reorganization of nucleosomes that occurs in conjunction with activation of genes for transcription.
- Numerous ATP-dependent chromatin remodeling complexes use energy provided by hydrolysis of ATP.
- All remodeling complexes contain a related ATPase catalytic subunit, and are grouped into subfamilies containing more closely related ATPase subunits.
- Remodeling complexes can alter, slide, or displace nucleosomes.
- Some remodeling complexes can exchange one histone for another in a nucleosome
histone acetyltransferase (HAT)
An enzyme (typically present in large complexes) that acetylates lysine residues in histones (or other proteins).
-Also known as lysine (K) acetyltransferase (KAT).
histone deacetylase (HDAC)
– Enzyme that removes acetyl groups from histones; may be associated with repressors of transcription. (closed conformation of DNA)
- Deacetylases are present in complexes with repressor activity.
-Deacetylation is associated with repression of gene activity.
Combinatorial control in eukaryotes
- GAL genes in Yeast
- Genes that are needed for eukaryotic cell to be able to use galactose if glucose is not available
Upstream activating sequence (UAS) in Yeast
- The equivalent in yeast of the enhancer in higher eukaryotes
- it is bound by transcriptional activator proteins.
GAL genes in Yeast
- Many genes scattered over many chromosomes – no operons like in bacteria – but still regulated coordinately by common set of proteins
- All GAL genes have similar promoters and are regulated by common set of proteins
5 levels of regulation by GAL genes in Yeast
- Chromatin opening (SWI/SNF, acetylation)
- Non-coding RNA transcripts
- UAS has both enhancer and Mig1 repressor binding sites
- GAL-specific induction system
- Catabolite repression
Activation by GAL genes in yeast
- GAL1/10 genes are positively regulated by the activator Gal4.
- GAL1/10 genes are negatively regulated by a noncoding RNA synthesized from a cryptic promoter that controls chromatin structure
GAL1 gene
- GAL1 is transcribed to the right- promoter region is 118 bp long and contains 4 upstream activator sites (UAS)
- GAL10 is transcribed in the opposite direction from the same control region
- UAS bind DNA-binding trans-activator protein made by GAL4 gene - GAL4p
- Transctivator GAL4p binds to UAS in front of many GAL genes
How to turn on GAL genes
- Need Gal4p – transcription activator – needed to bind to RNA polymerase to start transcription of genes so cell can use galactose
- involves additional proteins besides GAL4p including the inhibitor GAL80p, ligand sensor Gal3p, Mig1 which is present in nucleus dependent on phosphorylation and dependent on absence of glucose and Tup1 which binds to mig1 and blocks transcription as a repressor
Repression by GAL genes in yeast
- Gal4 is negatively regulated by Gal80.
- Gal80 is negatively regulated by Gal3, the ultimate positive regulator, which is activated by the inducer, galactose.
- Activated Gal4 recruits the machinery necessary to alter the chromatin and recruit RNA polymerase.
- Catabolite repression is mediated by a glucose-dependent protein kinase, Snf1.
GAL-specific induction system
GALACTOSE Absent:
- Gal80p made
Binds to Gal4p and blocks Gal4p from binding to RNA polymerase to help start transcription
GALACTOSE Present
- Galactose binds to Gal3p which binds to ATP
- Activated Gal3p binds to Gal80p, causes a conformational change in Gal80p so it no longer blocks Gal4p from binding to RNA polymerase
Basic helix- loop- helix motif
● Important for DNA binding and
protein dimerization
● One helix is for DNA binding
● Other helix is for protein - protein
binding → creating a dimer
Zinc finger motif
● Zn interacts with 4 Cys or 2 Cys
and 2 His
- Stabilizes the structure of
the protein
● Wind around DNA
Lactose Operon
Group of genes that produce enzymes that allow E.coli to use lactose as a carbon
source
● Controlled by both positive and negative control
Lacl gene has its own promoter and terminator
- End of the lacl region is adjacent to the lacZYA promoter, P
- Lacl produces a repressor that control whether or not RNA pol can bind to
promoter region and creates mRNA
● Transcription from P produces one polycistronic mRNA that has 3 translational
reading frames that produce proteins from the 3 genes that are transcribed as a
single mRNA (polycistronic mRNA)
1. lacZ
2. lacY
3. lacA
● Works through negative regulation
● Turns ON transcription (need inducer) → causes induction
- Allolactose is an environmental trigger signifying an abundance of lactose
- Allolactose will bind to the repressor and change its shape
- RNA pol will bind to the operator region instead and start transcription of
two genes
● Lactose permeates into the cell
through galactoside permease produced
by LacY gene
● Lac Z then produces β- galactoside
that cleaves the lactose molecule into
galactose & glucose by hydrolysis and
allolactose through isomerization
- Allolactose can act as inducer → turn on genes to create more β- galactoside
Part 1 of lactose operon
Operon with 3 genes
1. lacZ
2. lacY
3. lacA
● One promoter
● One operator sequence
- This where the repressor binds (lacO region)
Part 2 of lactose operon
● Gene lac l that produces the repressor protein
● Not regulated (lacl repressor is ON all the time)
- Once made can bind to region and dictates whether the rest of the
gene can be made
Part 3 of lactose operon
● Common gene that produces an activator protein
Lac Y gene
Produces galactoside permease
Lac Z gene
Produces β- galactoside
- Cleaves lactose and produced
allolactose → used as an inducer
- Can also cleave lactose into
galactose and glucose → used
as a C source
Arabinose operon
- Arabinose –a secondary sugar that can be metabolized and used for energy
- AraC – makes repressor for this operon – negative control
- CRP-cAMP – also needed for positive control
- Make repressor and turns the operon off, same idea, CRP and cAMP uses exactly the same activator as lactose.
Repressor (Ara C) can bind to three sites:
ara1, ara2 and araO2
When bound to araI1 and araO2, it causes a DNA loop and operon is can not be transcribed
When arabinose is present (inducer)
AraC binds to ara1 and ara2 and no loop is formed
and then CRP can bind to cAMP and help RNA polymerase bind and therefore transcription is started
Galactose Operon
Repressor does not stop RNA polymerase from binding but stops it from forming open complex
When CRP with cAMP binds to RNA polymerase, an open complex is formed and transcription is started
Separation of proteins
Use chromatography
* Ion exchange – based on charge
* Gel filtration – based on size
* Affinity resin – based on protein –
protein interaction
