Topic 8 (Transcription) Flashcards
Where in the eukaryotic cell does transcription happen?
Nucleus
Where in the eukaryotic cell does translation happen?
Cytoplasm
What are the major differences between DNA replication and RNA transcription?
DNA: copy the entire genome once an only once per cell cycle, and both strands serve as templates for new DNA synthesis
RNA: selectively copy only certain parts of the genome from one to multiple times and only one of the DNA strands serves as a template
The template strand is also called the:
Antisense or noncoding strand
The non-template strand is also called the:
Sense or coding strand
The RNA transcript dissociates from the template a few nucleotides behind the point of synthesis, which allows for:
Multiple transcriptional events of the same gene and translation to occur rapidly (prokaryotes)
RNA Pol:
Catalyzes RNA synthesis in the absence of primers
How do RNA Pol and DNA Pol differ in terms of proofreading? Why?
RNA Pol lacks extensive proofreading mechanisms (except its involvement with TCR); If RNA Pol makes a mistake, it is not severely detrimental to the cell
How many RNA Pol do eukaryotes have? Prokaryotes?
3; 1
Which subunits of RNA Pol are conserved among all organisms?
2 alpha and 2 beta subunits
RNA Pol I transcribes:
rRNA precursor (large RNA)
RNA Pol II transcribes:
mRNA (protein coding)
RNA Pol III transcribes:
tRNA and 5S rRNA
What are the phases of transcription?
- Initiation
- Elongation
- Termination
What is the +1 site?
It is the first nucleotide transcribed
True/False? The +1 site is always an A (start codon = AUG)
False. It depends on the 5’ UTR. AUG is the first translated codon
Describe the GENERAL steps of transcription initiation
- Formation of a closed complex (DNA is not melted yet) by binding Pol to the promoter
- Closed complex transformed into an open complex
- Initial transcribing complex makes the first 10 ribonucleotides
What element determines which DNA stretch will undergo transcription?
The promoter
What direction does transcription occur in?
5’-3’
The transcription bubble is an example of what?
An open complex
Describe the GENERAL steps of transcription elongation and termination
- Continual RNA synthesis
- Unwinds the DNA in front and reanneals it behind
- Emergence of the growing RNA from the template
- Proofreads
- Transcription stops and RNA product is released
Prokaryotic vs eukaryotic transcription initiation
Prokaryotic: only have 1 RNA Pol and one initiation factor (sigma)
Eukaryotic: 3 RNA Pol and requires several initiation factors for promoter-specific initiation
What sequences make up the core promoter for RNA Pol II from 5’ to 3’?
BRE, TATA, Inr, DPE
What protein binds BRE?
TFIIB
What protein binds TATA?
TBP
What protein binds Inr?
TFIID
What protein binds DPE?
TFIID
Which sequence contains the +1 site?
Inr
What is a core promoter?
Minimal sequence required for accurate transcription initiation in vitro
BRE stands for:
TFIIB recognition element
TATA stands for:
TATA box (element)
Inr stands for:
Initiator
DPE stands for:
Downstream promoter element
What is unique about DPE?
It is transcribed
TBP stands for:
TATA binding protein
What is contained upstream of the core promoter?
Regulatory sequences required for efficient transcription in vivo
Sequence elements upstream of the core promoter (6)
Promoter proximal elements
Upstream activator sequences
Enhancers
Silencers
Boundary elements
Insulators
How was the binding sequence of transcription factors (TFIIB, TBP, TFIID) discovered?
Each was added one at a time in vitro and transcription was measured to determine what order the TFs bound in
Transcription factors require these two domains:
DNA-binding domain and activation domain
What proteins is TBP associated with?
10 other TBP-associated factors (TAFs)
What is the function of TFIIB?
Binds the pre-initiation complex after TBP and may function in bridging between TATA-bound TBP and RNA Pol II. Also responsible for the directionality of transcription
How is TFIIB responsible for the directionality of transcription?
Binds upstream (5’) of TBP, so RNA Pol II is prevented from binding there and must bind downstream. Defines asymmetric assembly of the pre-initiation complex and unidirectional transcription
What is the function of TFIIF?
Recruited to the promoter with Pol II. This complex stabilizes the DNA-TBP-TFIIB complex and recruits TFIIE and TFIIH
What is the function of TFIIE?
Recruits and regulates TFIIH
What is the function of TFIIH?
Controls ATP-dependent transition of the pre-initiation complex to the open complex, phosphorylates Pol II C-terminal domain and causes promoter melting and escape. Also functions in NER
What are 5 examples of TAFs (TBP-associated factors)?
TFIIB, TFIID, TFIIF, TFIIE, TFIIH
What is the pre-initiation complex?
Protein complex containing Pol and general transcription factors
What part of the DNA does TBP bind?
Minor groove of the TATA element through its beta-sheet
What is unique about TBP associating with DNA?
Uses a beta-sheet to associate, while other TFs usually use alpha-helices to associate with the major groove
Describe the DNA conformational change when TBP binds
Minor groove is induced to be widened to almost flat, and the DNA is bent away from the TBP
Describe the steps to the assembly of the pre-initiation complex
- TFIID, TBP, and 11 TAFs recognize the TATA element. TBP binds TATA
- TFIIA and TFIIB bind upstream of the TATA
- Pol II with TFIIF binds
- TFIIE and TFIIH bind to complete the pre-initiation complex
- C-terminal domain of RNA Pol II becomes phosphorylated by TFIIH, promoter escape occurs, and transcription elongation begins
The C-terminal tail of RNA Pol II contains what?
Tyr residue repeats for phosphorylation
Why are additional proteins necessary for transcription initiation in vivo?
The DNA template in vivo is in chromatin form
What is the function of the activator proteins for transcription initiation?
Recruits Pol and stabilizes Pol:promoter interaction and binds to chromatin remodeling complexes
What is the function of the chromatin remodeler for transcription initiation?
Modifies nucleosome structure to facilitate transcription
What is the function of the HAT protein for transcription initiation?
Part of the chromatin remodeling complex, acetylates histone tails (loosens interaction)
What is the function of the mediator complex for transcription initiation?
Bridges the C-terminal domain of the Pol and the activator and regulates TFIIH
Which mediator complex subunit is required for Pol II transcription in vivo?
Med17
True/False? Mediator complexes have a similar shape and size to RNA Pol
False. Similar shape but larger size
How are mediator complexes organized?
Into modules (sub-complexes of multiple subunits
How does transcription elongation overcome the obstacle of chromatin?
FACT dimers
What does FACT stand for?
FAcilitates Chromatin Transcription
Describe the function of FACT on DNA that has already been transcribed
Restores H2A:H2B dimer to the existing hexamer
Describe the function of FACT on DNA yet to be transcribed
Disassembles H2A:H2B dimers from the octamer
What happens to initiation factors after initiation?
They dissociate from Pol II
Recruitment of elongation factors depends on what?
The phosphorylation state of the CTD (C-terminal domain) of RNA Pol II
What are elongation factors? Provide 2 examples
Factors that stimulate elongation; ELL protein family and TFIIS
What are the functions of TFIIS?
Increases rate of elongation by limiting Pol’s pause time and proofreads the new transcript
What are the RNA processing enzymes? How are they recruited?
5’ capping enzymes, splicing factors, and 3’ polyadenylation and cleavage factors; phosphorylation state of CTD
Describe the sequence of events following the phosphorylation of the CTD
- Capping enzyme is recruited to CTD tail
- Capping enzyme moves to 5’ end of new transcript, splicing machinery recruited to CTD
- 5’ capping enzyme dissociates, splicing machinery associates with the transcript, and polyadenylation and cleavage factors are recruited to the CTD
What stage of transcription is the capping enzyme recruited?
Promoter escape
What stage of transcription is splicing machinery recruited?
Elongation
Describe the steps of 5’ cap formation
- RNA triphosphatase removes the gamma phosphate at the 5’ end of the transcript
- Guanylyltransferase adds the GMP moiety to the terminal beta-phosphate
- Methyltransferase adds a methyl group to the guanine base (7-methylguanylate cap)
How many phosphates separate the first transcribed base and the 7-methylguanylate cap?
3 (beta and alpha from base and alpha from cap)
What is the function of the 5’cap?
Stabilizes the transcript and signals the transcript is correctly processed
Describe the steps of 3’ polyadenylation
- RNA Pol encounters and transcribes the poly-A signal (AAUAAA)
- Phosphorylated CTD recruits polyadenylation enzyme (CSPF and CstF)
- RNA Pol II transcribes until it falls off the template
- CSPF and CstF and other proteins cleave downstream of the poly-A signal, CSPF stays on the transcript
- Poly-A polymerase (PAP) adds ~200 adenines to the 3’ end of the poly-A signal
- Poly-A binding protein (PBP) coats the As to stabilize them and protect them from 3’-5’ exonuclease
What is the function of the poly-A tail?
Stabilizes mRNA and signals correct 3’ end processing
Describe the torpedo model of termination
- 5’-3’ exonuclease (Rat1/hXrn2) binds RNA Pol II
- Termination signal is transcribed and mRNA falls off while RNA Pol keeps transcribing
- exonuclease degrades the uncapped end of the newly synthesized RNA coming out of RNA Pol, which is faster than synthesis
- RNA Pol dissociates from the template
Describe the allosteric model of termination
- Termination signal is transcribed and mRNA falls off while RNA Pol keeps transcribing
- As RNA Pol transcribes, its processivity decreases as a result of a conformational change
- RNA Pol falls off, leaving the second RNA strand to be degraded
Why is RNA Pol I transcription so high?
There are many isoforms of rRNA that need to be transcribed by RNA Pol I
True/False? RNA Pol II is the only eukaryotic RNA Pol that is protein coding
True
Describe RNA Pol I transcription initiation
- UBF dimer binds upstream control element (UCE)
- SL1 (TBP + 3 TAFs) binds the core promoter upstream of +1
- Pol I binds
What is unique about RNA Pol III transcription?
The start site is upstream of the promoter
Why are the promoters internal to tRNA and 5S RNA?
Because they’re transcribed by RNA Pol III, their promoters are transcribed because the start site is upstream of the promoter
Describe RNA Pol III initiation
- TFIIIC binds Box A and B
- TFIIIB and TBP bind just upstream of the start site
- RNA Pol III is recruited to TFIIIB and TBP
- TFIIIC is displaced by RNA Pol III binding
What are the themes of eukaryotic gene regulation?
- transcription initiation is the primary point of gene regulation
- nucleosomes and their modifiers have a profound influence on gene expression
- transcription is controlled by activators and repressors
- regulation of RNA splicing is also important for gene expression
What is a promoter?
Region of DNA involved in pre-initiation complex binding
What is a regulatory binding site?
Binding site for different transcription factors
What is a regulatory sequence?
Entire collection of regulator binding sites for a given gene
What is an enhancer?
Tight cluster of regulatory binding sites that can affect long distances at either upstream or downstream of a gene
What is an upstream activating sequence (UAS)?
An enhancer in yeast only found upstream of a gene and usually close in proximity
What is an insulator?
Blocks promoter activation by binding activators at the enhancer
Describe the trend of regulatory sequences from bacteria to yeast to human genes
Increasing complexity. Humans use multiple regulatory elements both upstream and downstream from the promoter
Describe Gal4 structure
Has a distinct DNA-binding and activation domain (modular architecture), and often acts as a dimer
True/False? A different activator binds to each regulatory sequence, and all regulatory sites are bound at the same time for one gene
False. Not all regulatory sites need to be bound
What is GAL1?
A gene in yeast that is activated in the presence of galactose and absence of glucose by Gal4
Describe the UAS of GAL1
It’s made up of 4 regulatory sequences that each bind a dimer of Gal4
If the activating domain of Gal4 is removed, what happens to reporter gene transcription? What does this imply?
Reporter gene is not turned on but Gal4 still dimerizes; activation domain is required for eukaryotic transcription initiation
If the activating domain of Gal4 is hybridized with the DNA-binding domain of LexA, what happens? What does this imply?
The reporter gene controlled by LexA is turned on; eukaryotic transcription activators consist of a DNA binding domain and activation domain
Are both the DNA binding domain and activating domain needed for gene transcription in eukaryotes?
Yes
What does Y2H stand for?
Yeast two-hybrid assay
Describe how a Y2H works
Protein B is given an activating domain but no DNA binding domain. Protein A is given a DNA binding domain but no activating domain. Interactions between A and B allow for reporter gene transcription
What is a Y2H?
Protein-protein binding assay based on properties of yeast TFs widely used to search for binding proteins through a genetic screen
What are the types of DNA binding domains?
- helix-turn-helix motif
- zinc-containing DNA binding domains
- leucine zipper motif
- helix-loop-helix
What are the characteristics of activation domains?
- lack of defined motifs
- grouped by amino acid contents (rich in a certain amino acid/type of amino acid)
- may have “sticky” surfaces, often without single specificity for the protein it binds to
What is the helix-turn-helix motif? Where in the DNA does it bind?
Two alpha helices separated by a short turn; specific sequence within the major groove
Provide an example of a protein that uses the helix-turn-helix motif
Lambda repressor
What is a homeodomain protein? How does it interact with DNA and what organisms is it found in?
Three alpha helices recognize a specific sequence within the DNA major groove, as well as additional contact with the minor groove. Found in all eukaryotes
What is a zinc-containing domain? What is the function of the amino acid residues that associate with the zinc?
Recognition helix and beta sheets associate with Zn2+ ion through 2 His and 3 Cys residues, which stabilize the ion and allow for proper DNA binding
What is a leucine-zipper motif? Where in the DNA does it bind?
Dimerization of two alpha helices that have a Leu rich sequence (interact with major groove) and a coiled-coil domain (interact with each other for dimerization)
What is a helix-loop-helix domain? Where does it associate with DNA?
A dimer consisting of one short and one long alpha helix each, where the short helices interact with each other and the long helices interact with the major groove
Describe chromatin immunoprecipitation (ChIP)
Proteins crosslinked with the promoter with formaldehyde are recognized by an antibody specific to that protein. The antibody is then pulled down with an agarose or magnetic bead to isolate this protein + DNA from other cellular components. The protein + antibody are removed, leaving the purified DNA to be used in a microarray or to be sequenced following PCR
What are the major functions of transcription activators?
Protein recruitment and work cooperatively and synergistically to promote combinatorial control
List the ways in which transcription activators recruit proteins
- recruit other TFs
- recruit nucleosome modifiers
- indirectly recruits RNA Pols
- recruits factors needed for initiation and elongation
What contributions do transcription activator’s cooperative and synergistic work make?
Complexity and diversity in eukaryotes
What does it mean that the effects of TA’s are additive?
The more interactions with activators, the stronger the stimulation of transcription
What proteins are recruited directly and indirectly to the promoter by activators, respectively?
TFIID and mediator, RNA Pol II (through mediator)
What proteins are recruited by activators for nucleosomal modification?
Histone acetylase (HAT), which increases expression, or the chromatin-remodeling complex, which uses ATP to physically move nucleosomes to expose DNA for transcription
If an insulator is placed between an enhancer and a promoter, what happens?
Inhibition of enhancing effects
If there is an enhancer being blocked by an insulator, but the promoter is still on, what’s happening?
An enhancer that isn’t restricted by the insulator is enhancing that promoter
True/False? Insulators can prevent enhancer effects on any promoter in proximity (upstream or downstream of the enhancer) as long as one side of the promoter is blocked
False. If one side of the enhancer is blocked by an insulator, then the promotor on the same side is inhibited, but the promoter on the other side is not
Describe the looped-out model for enhancer function
An enhancer from a distant sequence on the same chromosome may loop around and associate with the promoter through a TF
Why are enhancers distance and orientation independent?
Looped-out model for enhancer function
What does the locus control region (LCR) regulate?
Expression of a cluster of 5 hemoglobin genes in humans
What is special about the 5 hemoglobin genes in humans regulated by LCR?
They show temporal and spatial regulation in their expression
What is the homologous structure in mice of LCR?
GCR (global control region)
How is LCR hypothesized to work?
Somehow works through chromatin structure
Describe the synergistic binding of activators
- different activators can recruit a single protein by touching different parts of it
- the effect of activators working together is greater than the sum of the individuals alone
- synergy serves as a checkpoint to ensure proper signals are received
Describe direct interactions between activators
Cooperative binding through direct interactions
Describe indirect interactions between activators
Both proteins interact with a common protein but not each other
Provide and explain a general example of an indirect interaction between activators
Activator A either recruits a nucleosome remodeling complex or unwinds the DNA from the nucleosome itself to reveal a binding site for activator B
Describe a specific example of signal integration (indirect interaction between activators) in yeast
HO gene expression in yeast; SWI5 (only active in mother cells) recruits the chromatin remodeling complex and histone acetylase to loosen DNA-nucleosome interactions at the SBF binding site. SBF binds to its now open binding site (only at the correct cell cycle stage)
Describe a specific example of signal integration (indirect interaction between activators) in humans
Human beta-interferon gene; upon infection, HMGA1 binds to the minor groove to help the enhanceosome assembly by keeping the enhancer straight. Activators (NF-KB, IRF, Jun/ATF) bind to enhancer and INF-beta gene is turned on
List the ways transcription repressors can work
- overlap with DNA binding site (prokaryotes, competitive)
- inhibition of transcription machinery by interfering with activators, mediators, or RNA Pols (direct repression)
- bind to activating domain on TFs (inhibition)
- recruit histone modifiers to further compact the chromatin structure (indirect inhibition)
What is a common regulator?
A TF that may activate more than one gene
What are the MAT locus, gene regulatory proteins, and target genes of a haploid a cell?
MAT: a
proteins: a1 and Mcm1
target genes: aSG on (Mcm1 bound), alphaSG off (nothing bound) hSG on (nothing bound)
What are the MAT locus, gene regulatory proteins, and target genes of a haploid alpha cell?
MAT: alpha
proteins: alpha1, alpha 2, Mcm1
target genes: aSG off (Mcm1 + 2 alpha2), alphaSG on (alpha 1 +Mcm1), hSG on (nothing bound)
What are the MAT locus, gene regulatory proteins, and target genes of a diploid a/alpha cell?
MAT: both a and alpha
proteins: a1, Mcm1, alpha 2 (alpha1 not transcribed)
target genes: aSG off (Mcm1 + 2 alpha2), alphaSG off (none bound), hSG off (alpha2 and a1 bound)
alpha2 in yeast sex determination is what kind of protein?
Repressor
Why does alphaSG require both Mcm1 and alpha1 to be turned on?
If alphaSG didn’t require alpha1, it would be turned on in a cells because Mcm1 is present in both
Describe the JAK/STAT pathway
- Ligand binds a receptor tyrosine kinase (RTK)
- RTK dimerizes, phosphorylates itself and other monomer
- STAT uses SH2 domain to recognize phosphorylated RTK and gets phosphorylated by RTK
- STAT dimerizes and relocates to nucleus to act as a TF
Describe the MAPK pathway. What is Jun involved in?
- Ligand binds RTK autophosphorylation
- Grb2/SOS bind RTK and nearby Ras
- SOS switches out GDP to GTP on Ras (now active)
- Ras phosphorylates MAPKKK
- MAPKKK phosphorylates MAPKK
- MAPKK phosphorylates MAPK
- MAPK phosphorylates Jun/Fos
- Jun/Fos are transcription activators
Jun is an activator for the INF-beta gene (card 134)
Where is transcriptional silencing most common?
Centromeres, telomeres, genes used in other types of cells
What are the yeast silence information regulator (SIR) proteins? Where are they found?
Rap1 (DNA-binding protein), Sir2 (histone deacetylase), Sir3, Sir4; telomeres
List the mechanisms to block the spreading of silenced regions of DNA
- insulator elements can block the spread of histone modification
- histone methyltransferases may repress spreading of Sir2-mediated silencing
- methylation of histone H3 tail
Methylation of H3K4 results in:
Increased gene expression
Methylation of H3K9 results in:
Decreased gene expression
What is a CpG island?
A cluster of C’s followed by G’s that are methylated to silence gene expression
What is MeCP2?
A protein that binds methylated DNA in mammals that recruits histone modifiers and nucleosome remodelers to completely turn off genes by making chromatin inaccessible
True/False? Gene silencing by methylation of CpG islands completely suppresses transcription
False.
Describe what happens on the maternal chromosome in maternal imprinting of the Igf2 gene
H19 is transcribed, and the insulator (CTCF) blocks the enhancer from stimulating transcription of Igf2 (not methylated)
Describe what happens on the paternal chromosome in maternal imprinting of the Igf2 gene
H19 and the insulator are methylated, which prevents the insulator from binding, which stimulates transcription of Igf2
Which chromosome (methylated or unmethylated) expresses the Igf2 gene?
Methylated
What is uniparental disomy/Beckwith-Wiedermann syndrome?
Excessive expression of paternal genes/loss of maternal contribution of genes on chromosome 11 results in increased Igf2 transcription and no active copy of H19 (inhibits cell proliferation). It is an overgrowth disorder characterized by overgrowth, enlarged tongue, severe hypoglycemia, midline abdominal wall defects, and increased risk of childhood cancers
How are patterns of DNA methylation passed onto new strands of DNA after replication?
CpG is palindromic, so upon replication, any hemimethylated CpGs are methylated by a maintenance methylase
