Chapter 6: 6.4 Eukaryotic Transcription Flashcards
How is eukaryotic DNA different from prokaryotes?
Packaged into chromating
* In condensed (default) state results in apromoter that is inaccessible to RNA polymerase
In eukaryotes, when can transcription occur?
Only when DNA is decondensed and dissociated from the histones around which it is wound
Can transcription and translation occur at the same time in prokaryotes? Eukaryotes?
Yes, but they cannot occur at the same time in eukaryotes because eukaryotes have a separate compartment for the DNA (nucleus)
Prokaryotic cells have polycistronic mRNA, what does that mean?
Genes with common function are arranged linearly and are transcribed together on a single mRNA
Eukaryotic mRNA is always monocistronic, what does this mean?
One mRNA = One protein
How do gene locations differ between prokaryotes and eukaryotes?
Eukaryotic cells: Genes with common functions can be scattered throughout the genome, on different chromosomes and can therefore be transcribed and regulated separately from one another
How many types of RNA polymerases do Eukaryotes have?
3
1. RNAP I: Transcribes rRNA
2. RNAP II: Transcribes mRNA
3. RNAP III: Transcribes tRNA
How does the mRNA differ in eukaryotes to prokaryotes?
mRNA requires extensive modification before being translated into protein in Eukaryotes
What are key differences in transcription between prokaryotes and eukaryotes?
(8 points)
- Chromatin in eukaryotes
- Transcription and translation cannot occur simultaneously in eukaryotes
- Monocistronic mRNA in Eukaryotes vs. Polycistronic mRNA in Prokaryotes
- 3 types of RNA polymerase in eukaryotes
- mRNA requires modification before being translated in Eukaryotes
- Consensus sequence is TATA box in eukaryotes
- Eukaryotes require several transcription factors (TFs) instead of a Sigma
- Termination can be different in eukaryotes
True or False:
Promoters are more complex in eukaryotes but they do not have consensus sequences
False, they are more complex AND they have promoter sequences
What is the consensus sequence in an eukaryote?
TATA box
* Located about ~30 nucleotides upstream of the transcriptional start site
How is transcription started in eukaryotes?
Requires help of transcription factors
* Binds to DNA and start transcribing
True or False:
The elongation and termination process are same between prokaryotes and eukaryotes
Not exactly, the elongation process is identical but the termination process can be different
In eukaryotes:
What is the equivalent to sigma proteins in prokaryotic transcription?
Basal transcription factors
Define:
TATA-binding protein (TBP)
A basal transcription factor
* Binds to the TATA box in the promoter of eukaryotic genes
* RNA polymerase is recruited following TBP binding
Besides TBP, what do other transcription factors do in eukaryotes?
Bind to different areas within the gene and interact with the polymerase to initiate transcription
True or False:
Enhancers can even be located far away from the genes they affect
True
What is the responsibility of transcription factors?
Help coordinate gene expression
* Genes lcoated on different chrosomes can be controlled simultaneously through the use of the same transcription factor
Prokaryotic vs. Eukaryotic Transcription:
Gene organization
Prokaryotes
* Genes with common function are arranged linearly and are transcribed together (polycistronic)
Eukaryotes
* Genes with common functions can be scattered throughout the genome (monocistronic)
Prokaryotic vs. Eukaryotic Transcription:
Location of Transcription
Prokaryotes
* Transcription and translation happens in the same compartment
Eukaryotes
* Transcription occurs in the nucleus and translation happens in the cytoplasm
Prokaryotic vs. Eukaryotic Transcription:
RNA Transcript Processing
Prokaryotes
* mRNA is transcribed directly from DNA
Eukaryotes
* RNA transcripts must go through several processing steps before becoming mRNA
What steps must RNA transcripts go through to become mRNA in eukaryotes?
- 5’ and 3’ UTRs are retained
- 5’ cap structure: (7-methlylguanylate) is added to the 5’ end of the RNA transcript
- 3’ poly-A tail: Polyadenylation of the 3’ end of the RNA transcript
- Gene splicing: Intron excision and exon ligation
Prokaryotic vs. Eukaryotic Transcription:
Transcriptional regulation
Prokaryotes
* Sigma protein
Eukaryotes
* Transcription factors and enhancers
Prokaryotic vs. Eukaryotic Transcription:
RNA Polymerases
Prokaryotes
* One RNA polymerase
Eukaryotes
* Three RNA polymerases
Where are the 3 RNA polymerases located and what do they do in the eukaryote?
- RNA Pol I: rRNA (located in the nucleolus)
- RNA Pol II: mRNA (located in the nucleoplasm)
- RNA Pol III: tRNA (located in the nuceloplasm)
How do scientists determine which of the three eukaryotic polymerases are present?
By determining their sensitivity to α-amanitin
State:
α-amanitin sensitivity of the different polymerases in eukaryotes
- RNAP II activity is very sensitive to α-amanitin, cannot transcribe RNA in its presence
- RNAP I and RNAP III can still transcribe RNA in presence of α-amanitin
Eukaryotic polymerases can be purified by ————– (—-, — or ——– ——–)
- Chromatography
- Size
- Ion
- Antibody affinity
Transcribed RNA can be detected by what?
Northern Blot
What is the CTD?
RNAP II Carboxy-Terminal Domain
* Made up of Tyr-Ser-Pro repeats
How many Tyr-Ser-Pro repeats are in human CTD?
52
During the initiation of transcription, what is the CTD like?
Unphosphorylated
When does the CTD become phosphorylated?
As the polymerase begins the elongation phase of transcription
What can bind to the CTD depending on it’s phosphorylation state?
(3 points)
RNA processing proteins
* Capping
* Elongation
* Splicing factors
Describe:
Transcription Factor II (TFII)
Made up of multiple subunits which help position RNAP II at the start site and initiate transcription
Define:
TFIID
Consists of:
* TATA box Binding Protein (TBP)
* 13 other TBP-Associated Factors (TAFs)
TFIID forms a complex with —- - and —– and the ——–
- TFIIA
- TFIIB
- TATA-box
Define:
TFIIF
Binds to RNAP II
Define:
TFIIE
Binds and creates a site for TFIIH to bind
Define:
TFIIH
Has helicase activity which unwinds the DNA
* After transcription has been initiated, has kinase activity which phosphorylates the CTD of RNAPIII
List the process:
General Transcription Factors (GTFs) attaches
(5 points)
- TFIID forms complex with TFIIA and TFIIB and the TATA-box
- TFIIF and RNAP II then bind
- TFIIE binds and creates a site for TFIIH to bind
- TFIIH has helicase activity which unwinds the DNA
What are the general transcription factors required for initiation?
- Phosphorylation of the RNAP II carboxy-terminal domain (CTD)
- General Transcription Factors (GTFs)
What do transcriptional activators do?
Activate transcription by binding to control regions on the DNA
What are the control regions on the DNA?
- Enhancer sites
- Promoters
- Initiator sites etc.
What do transcriptional activators have?
- DNA binding domain
- Activation domain
In a transcriptional activator:
Describe a DNA binding domain
- Zinc finger domains (C2H2 or C4)
- Leucine zipper
- Basic Helix-Loop-Helix
In a transcriptional activator:
Describe an Activation domain
Short structures containing high percentage of one or two amino acids (e.g. Asp, Glu, Gln, Pro, Ser or Thr)
What is the function of Activation domains?
Facilitates protein-protein interactions
* May have a random structure until interaction with a co-activator protein
Why are GTFs a unique set of transcription factors?
They are required to initiate almost all RNAP II activity at the promoter
Are GTFs considered to be activators of transcription in the same way as DNA binding domain/activation domains?
No
What do Transcriptional Repressors do?
Repress transcription by binding to control regions on the DNA
What do Transcriptional Repressors have?
- DNA binding domain
- Repression domain
In a transcriptional repressor:
Describe a Repression Domain
Short structures (<15 amino acids)
What is the function of Repression Domain?
Facilitates protein-protein interactions
* May need to bind to co-repressor proteins to silence transcription
Control regions on DNA often have multiple sites where…
Transcriptional activators AND repressors can bind
Depending on which type of transcription factor binds to the control region, what could happen?
Gene transcription can be activated or repressed
Define:
Heterodimers
Combinations of different transcriptions binding together to interact with different DNA sequences
Define:
Inhibitory factors
Can block the activity of some transcription factor DNA binding domains
Define:
Cooperative Binding
Unrelated transcription factors binding to neighboring sites
How does a finite set of transcription factors control the expression of so many genes?
- Heterodimers
- Inhibitory factors
- Cooperative Binding
DNA replication and transcription is dependent on the physical state of what?
Chromatin
Define:
Heterochromatin
The tightly packed, condensed form of chromatin
* Transcriptionally “silent”
Define:
Euchromatin
More open, de-condensed form of chromatin
* Transcriptionally “active”
In Post-Translational Histone Modification:
What tails of the histone proteins can be modified?
- C-terminus tails
- N-terminus tails
What are the most widely-studied post-translational histone modifications?
- Mono-, di- or tri-methylation on Lysine (K) or Arginine (R)
- Acetylation on Lysine (K)
- Phosphorylation on Serine (S) or Threonine (T)
True or False:
Post-translational Histone Modifications correlate to chromatin state
Not exactly, only some of these modifications correlate to chromatin state
Give examples:
Modifications that correlate to chromatin state
- H3Kme3: Causes chromatin to decondense (euchromatin)
- H3Kme2: Causes chromatin to condense (heterochromatin)
- Acetylation of Lysine: Usually correlates with euchromain
- Deacetylation of Lysine: Usually correlates with heterochromatin
What kind of processes are heterochromatin/euchromatin?
Positive Feedback Loop
In heterochromatin, what is recruited?
Methyl transferases
In euchromatin, what is recruited?
Acetylases
In heterochromatin, a ———– mark can recruit proteins (e.g. —)
- Methylation
- HP1
In euchromatin, an ———- mark can recruit proteins (e.g. ———– ——–)
- Acetylation
- Bromodomain proteins
What happens to Eukaryotic mRNA primary transcripts after transcription?
Have to undergo additional processing (mRNA processing)
What are the 2 important processes in mRNA processes?
- 5’ Capping
- 3’ Polyadenylation
- Splicing
Describe:
5’ Capping
The 5’ end of the mRNA is capped with a 7-methylguanosine
In 5’ Capping, what is the 5’ end of the mRNA capped with?
7-methylguanosine
What does 5’ Capping do?
- Provides stabilization
- Allows export from the nucleus
- Acts as a signal to intiate translation
Describe:
3’ Polyadenylation
100-300 adenine (A) bases are added to the 3’ end of the mRNA
In eukaryotes, how does termination work on the 3’ end?
Has a AAUAAA sequence
* Once transcription is terminated, the mRNA transcript is cleaved after this sequence
What does 3’ Polyadenylation do?
- Provides stabilization
- Export from the nucleus
- Translation
State:
How to remember the difference between exons and introns
EXons are EXpressed, INtrons are IN between
What is splicing carried out by?
Spliceosomes
What are snRNPs? What are they comprised of?
Small Nuclear RiboNucleoprotein Particles, comprised of:
1. snRNAs, which base pair with the pre-mRNA
2. Proteins
What does snRNA stand for?
Small nuclear RNAs
* U-rich
What types of snRNPs are there?
U1, U2, U4, U5, U6
Splicing occurs through what reactions?
Transesterification reactions
Describe:
The 2 transesterification reactions in splicing
- The ester bond between the intron’s 5’ phosphorus and the first exon’s 3’ oxygen is EXCHANGED with an ester bond on the 2’ oxygen of the branch point A
- The ester bond between the second exon’s 5’ phosphorus and the intron’s 3’ oxygen is EXCHANGED with an ester bond with the first exon’s 3’ oxygen
Abnormal splicing can lead to…
Disease
Define:
Cryptic splice sites
Point mutations destroy normal splice sites, which activates “hidden” splice sites within the intron
What do cryptic splice sites usually result in?
An extended exon
Define:
Exon skipping
Point mutations result in an entire exon being spliced out with the introns
Define:
New splice sites
Point mutation results in a new splice site within in the intron so part of the intron is expressed with the exons
