Fergus 1: Control of Gene Expression - some AI Flashcards
How can you know a gene is being expressed?
You cannot assume agene is working until you have an active protein product
i.e. gene expression is not just transcription of a gene
Give an example of when a gene is transcripted but not expressed
(3)
The mutation of delta f508 gene in CF
This results in a protein which has a fairly active ion channel
However the proteinas gets stuck in the golgi apparatus on its way to the cell membrane
Why do we need to regulate gene expression?
All cells in the human body have the same genome
Yet liver cells are distinctly different from cardiac cells
Gene expression is regulated to allow for the differentiation of cells
What does the mRNA from any cell represent?
Represents only the genes that are active in that cell at that moment in time
Talk about the expression of genes in Prader-Willi syndrome, and what problems arise in diagnosis?
(4)
Prader willi syndrome is caused by mutation of genes on chromosome 15
The gene is found on both copies of chromosome (maternal and paternal) but it is only expressed on the paternal gene
Therefore if you have paternal deletion you will have the syndrome -> you still have the gene but its not active
This can cause problems in detecting the syndrome as regular PCR will still detect the gene -> we need to look at the RNA instead to detect the syndrome
What is one reason why you might have transcription but no translation of a protein
mRNA can be attacked by miRNA (micrornase)
micro-RNAses can cause degradation of rna or failure to translate the rna
What are the basic steps in gene expression
DNA
Transcription by RNA polymerase II
hnRNA
Post transcriptional processing
mRNA
miRNAs and siRNAs
Translation
Protein
Post-translational modification
Protein product
What are three examples of post transcriptional processing?
Capping
Processing
Polyadenylation
Give two examples of RNases that act on mRNA
miRNAs
siRNAs
What are siRNAs?
Small interfering RNA
Also known as short interfering RNA or silencing RNA
What is capping?
(3)
The enzymatic modification of the 5’ end of mRNA
This protects the strand from degradation
A 7-methylguanosine (modified guanine) is added to thr 5’ end
What is polyadenylation?
The addition of a string of As
Addition of a poly (A) tail to the 3’ end
What is splicing?
The removal of any introns
Happens after transcription
What is RNA called before it has undergone post transcriptional processinf?
hnRNA or heterogeneous nuclear RNA
What is hnRNA?
Heterogeneous nuclear RNA
Is an immature form of mRNA which has yet to undergo post transccriptional processing such as splingin, capping and polyadenylation
i.e. it still contains introns and is not protected from degradation
What is the point of post transcriptional processing?
To make a chain of only exons
Which is protected from degradation
What signals the gene exoression?
Extracellular signals switch on genes
This then signals decompaction of DNA for transcription to occur
What enzyme is responsible for unwinding DNA?
Helicase enzyme
What is the role of RNA polymerase II
This transcribes HnRNA
What are the different levels of control over gene expression?
ALteration of chromatin structure
Epigenetic
Initiation of transcription by RNA pol II
Post transcriptional processing
Transport to cytoplasm
mRNA stability and degradation
Translation of RNA at ribosome
Post translational processing
How is gene expression regulated at a chromatin level
(3)
Most of DNA in a cell is highly compacted
In this form DNA is not available for transcription as helicase enzymes cannot get near DNA etc
The gene has to be made available for transcription
How is gene expression regulated on an epigenetic level?
(5)
Associated with chemical modifcation of DNA and of the proteins associated with DNA
Methylation especially of the cpg island
methylation of DNA impairs transcription
Genes not in use tend to be methylated
Abberant methylation of incorrect genes and acetylation of incorrect genes seen in cancer
Talk about the role of miRNAse in gene regulation
30% of human genes are regulated by micRNAse
Cancers tend to have disregulation of micRNAse
These bind to mRNa and either prevent it from being translated or they degrade it -> either ay we dont get a protein
How is gene expression controlled on a translational level
mRNA is translated to a protein which is often times not active -> protein needs activation
Transport, phosphorylation, glucosylation are all examples of post-translational processing needed to activate a protein
How is gene expression controlled on a translational level
mRNA is translated to a protein which is often times not active -> protein needs activation
Transport, phosphorylation, glucosylation are all examples of post-translational processing needed to activate a protein
What are the three differnet types of control on gene expression?
Developmental
Tissue specific
Environmental
What is meant by developmental contro over gene expression?
Temporal gene regulation
Gene regulation necessary for us to develop i.e. how do we get from a single cell to a human
Involves the switching on of genes to allow for cell differentiation and tissue development etc
Genes turned on due to a developmental need
What is meant by spatial control over gene regulation
Growth hormone produced only by cells in the pituitary gland
What signals are in place to control this -> the signals that only exist in these cells in this organ
What signals cause abberent expressio in disease states
Genes turned on due to their location
What is meant by environmental control of gene expression?
If you ingest heavy metals -> metallothionine genes switch on to deal with the heavy metals
i.e. genes only turned on because of external environmental factors
What are the different levels of chromatin structure?
(5)
Condensed scaffold-associated form
Extended scaffold-associated form
30nm chromatin fiber of packed nucleosomes
‘Beads-on-a-string’ form of chromatin
Short region of DNA double helix
At what chromatin levels is transcription possible, i.e. at what levels of unraveling can the helicase enzyme work
‘Beads-on-astring- form of chromatin
Short region of DNA double helix
What is meant by the ‘bead-on-a-string’ structure?
Nucleosomes
An optimer of histone (8 histones brought together)
DNA wraps around each histone to create the bead on a string shape
What forms of chromatin are you most likely to see in the cell?
Very rarely see chromosome in metaphase (traditional look of a chromosoem you imagine) -> very little gene expression possible in this form so only seen when cell is dividing
30nanometer chromatin fibre seen much more commonly but transcription still not possible in this form -> vast majority in this form
Talk about gene expression in Huntingtons disease
Huntingtons disease is a dominant disease - only need one copy of mutated chromosome
Mutation of chromosome 4
If you inherit one copy of the mutated gene you will suffer from the disease as the gene is pathogenic
Explain why some genetics are recessive and others are dominant
Mutations in dominant conditions tend to be pathogenic
Mutations in recessive conditions tend to be loss of function mutations -> hence why two copies of the mutation are required for the gene as you will still produce some functional protein
Give two examples of X linked disorders
Fragile X syndrome
Duchenn muscular dystrophy
Explain why X linked disorders affect only males
Males only have one copy of X chromosome while females have two
Males get full affect of any mutation on X chromosome while females can just use their second copy
What happens in expression in Downs Syndrome?
(3)
Extra opy of chromosome 21
Symptoms occus due to too much protein from genes on chromosome 21
Genes in a certain region of 21 are directly responsible
What is epigenetics?
(4)
It means above or in addition to genetic
Non-sequence dependant inheritence
Responsible for dfferentiation i.e. makes certain genes available for transcription
The reason why identical twins can still be different
Give an example of where epigenetics is important
(2)
When a muscle stem cell divides it will only ever make muscle cells even though muscle stem cells are similar to other stem cells
Every one of these cells have the same DNA so how do these cells know what genes should be active -> epigenetics
Give an example of where epigenetics is important
(2)
When a muscle stem cell divides it will only ever make muscle cells even though muscle stem cells are similar to other stem cells
Every one of these cells have the same DNA so how do these cells know what genes should be active -> epigenetics
Give some examples of the work of epigenetics
(3)
Identical twins with different natural hair colour
A single individual with two different eye colours
An identical twin liter mate with different coat colours
What was the Duke University carried out on clone mice to prove the effects of epigenetics?
(4)
Clone mice with identical genomes
Blastocysts were implanted into two different mice mothers
The mice mothers were fed different diets
The mice babies came out with completely different appearances
In what four ways are epigenetics controlled?
Chromatin structure - compacted vs uncompacted
Chemical modification of chromatic - methylation
Alternative splicing of RNA
RNA interference via miRNA and siRNA
Explain how we contol genetics through methylation
Via acetylation of histones and methylation of histones
Why is there a need for alternative splicing?
(3)
Most genes in the human genome produce more than one protein, because of this they dont need to use all exons everytime the gene is transcribed i.e. they only need some
e.g. Might use exon 1, 2, 3, and 4 for one protein but then might use exon 2, 3, and 5 for another protein
Its still the sam gene but can just have different products e.g. DMD gene
Why is there a need for alternative splicing?
(3)
Most genes in the human genome produce more than one protein, because of this they dont need to use all exons everytime the gene is transcribed i.e. they only need some
e.g. Might use exon 1, 2, 3, and 4 for one protein but then might use exon 2, 3, and 5 for another protein
Its still the sam gene but can just have different products
What does the work of miRNA or siRNA do?
It results in protein product
What does the work of miRNA or siRNA do?
It results in protein product
How does methylation control DNA transcription in an active cell vs an inactive cell?
Active cell:
- Open, active chromatin tends to be unmethylated (especially at promoter)
- The Histones of the chromatin, particularly H3 tend to be acetylated
Inactive cell
- Condensed chromatin, chromatin methylated (including at promoter)
- deacetylated/unacetylated histones
Talk about methylation of cytosines as a form of gene control
Sequence of Cs and Gs knon as CPG island
Cs and Gs are subject to methylation which inactivates DNA
Cs and Gs on both sides of strand are methylated
Give an example of a condition whereby CpG methylation is important
In fragile X the FMR1 gene is mutated to repeat CCGs
Mutations of over 200 CCG repeats results in methylation of the promoter and thus failure of the gene
This causes male intellectual disability
Give a basic description of the structure of histones, how do they compact DNA
A histone is a protein that provides strucutural support for DNA double helix
2 copies of histone A, B, C and D come together to make an optimer i.e. 8 histones make an optimer
DNA double helix wraps around the histone optimer
Each histone has a tail which extends out past the DNA double helix
How are histone molecules involved in gene control
Modification of the tails, particuarly H3 result in activation/inactivation of DNA -> H4 is often involved as well
H3 Lysine 4 methylation results in activation
H3 Lysine 9 methylation results in chromatin condensation which is associated with transcriptional repression
Acetylation of Lysine 9 activates transcription
How is H4 involved in gene controll?
Methylation of lysine 16 has transcriptional activation
How is H4 involved in gene controll?
Methylation of lysine 16 has transcriptional activation
What percentage of the human genome is regulated by methylation, how do we know this?
(3)
If a gene has a CPG island in its promoter it is controlled by methylation -> think of H3 and H4
60% of genes are have CPG islands
The remaining 40% have TATA sequences
How can methylation prevent transcription?
Methylation can prevent trascription complex from binding to a downstream to a promoter
i.e. if CpG island is methylated upstream it can prevent transcription complex from binding to a TATTA sequence downstream
-> this is because methylation of CpG is associated with chromatin condensation
??? im not sure on this
Why do genes require a promoter sequence such as GC or TATTA?
This is where RNA polymerase II will bind
What does an enhancer sequnce do and where are they found?
These modulate the rate of transcription
They are found throughout a sequence even in introns
What gene in the human genome has the most exons?
The Titin gene (TTN)
It has over 363 exons
Most conditions are caused by mutations in exons, give an example of a conditions whereby there is mutations within introns?
In Friedreichs ataxia
There is a mutation in intron 1 of the FXN gene
This results in expansion of a GAA-TC repeat tract which leadds to an mRNA deficit
Failure to transcribe and make mRNA
What are the four core promoter elements of non CpG island promoters?
BRE
TATA box
Inr/initiator
DPE/ downstream core promoter element
What are the four core promoter elements of non CpG island promoters?
BRE
TATA box
Inr/initiator
DPE/ downstream core promoter element
What is BRE?
TFIIB recognition element
Its found commonly enough and found near to the TATA sequence
GGG/CCA followed by CGCC
What is the sequence of a TATA box
There is some variability:
TATA followed by (A/T)A(A/T)(A/G)
What is the Inr?
An initator sequence
There is lots of variability in this sequence
What is DPE?
Downstream core promoter element
Usually found in the first exon
Lots of variation again in this sequence
What was the first hypothesis put forward on promoter sequences?
That the 5’ flanking sequence contributes to the initiation of transcription of genes
Give four examples of promoter sequences
TATA box
CAAT box
GC box
Oct site
Talk a little about the variation that occurs in the TATA box
There is some variation in all bases but its usually TATA followed by some sequence of Ts and As
100% of the time there is a T in the 3rd position
What is an example of a model promoter that we used to prove TATA was involed in transcription?
AMLP
Adenovirus major late promoter
But any TATAA containing sequence could be used
Give three examples of reporter genes we could use to prove TATA was involed in transcription?
Chloramphenicol acetyl transferase (CAT)
Luciferase
B Galactosidase
How to prove TATA was involed in transcription?
We connected TATAA to a gene whos product is easy to detect and put it into a cell that doesnt usually produce this produce
We then detected either the RNA or the product protein to prove our hypothesis
How does the CAT/Chloramphenicol acetyl transferase assay work to prove TATA was involed in transcription?
(4
We used the gene for chloramphenicol
We used promoters specific for chloramphenicol
Chloramphenicol protein produced
We then converted this to chloramphenicol acetyltransferase for easy detection
How does the B Galactosidase reaction work?
It uses the same method to prove TATAA through CAT except it produces light which is measured
How does the B Galactosidase reaction work?
It uses the same method to prove TATAA through CAT except it produces light which is measured colourimetrically
How does the luciferase assay work to prove TATA
It produces light which is measured
How does the luciferase assay work to prove TATA
It produces light which is measured
When using a bacterial plasmid to make a eukaryotic protein, what must be added into the plasmid?
You must add in a synthetic poly(A) signal to ensure the polyadenylation of the gene
Mammalian genes are polyadenylated while bacterial genes are not -> gene will not be translated if not polyadenylated
How would you detect the RNA of the CAT asay?
Reverse-trannscription PCR
What are the steps of reverse Pcr
Transfect construct
Isolate mRNA from transfected cells
Reverse transcribe RNA using reverse transcriptase
This gives us cDNA (for all genes)
PCR amplify using specific primers for CAT this cDNA to give us multiply copies
If we get a product then our assay has worked - Very high sensitivity
What are the steps in a northern blot?
Northern Blot
Transfect construct
Isolate RNA from transfected cells
Run on agarose gel first
Transfer to nitrocellulose membrane
Apply/Probe membrane with 32P labelled CAT probe
Detect presence /absence of signal/radioactive probe by applying an xray film and then developing it
Low Sensitivity
What are the steps in a northern blot?
Northern Blot
Transfect construct
Isolate RNA from transfected cells
Run on agarose gel first
Transfer to nitrocellulose membrane
Apply/Probe membrane with 32P labelled CAT probe
Detect presence /absence of signal/radioactive probe by applying an xray film and then developing it
Low Sensitivity
What are the steps to the nuclease protection assay
Transfect cell line with reporter construct
Isolate RNA from transfected cells (single stranded)
Hybridise with radiolabelled CAT probe -> forms double strands where binding occurs
Treat hybridisation mix with Sl nuclease -> degrades all single stranded molecules
Electrophorese and autoradiograph
Only gene of interest remains
Medium Sensitivity
What are the steps to the nuclease protection assay
Transfect cell line with reporter construct
Isolate RNA from transfected cells (single stranded)
Hybridise with radiolabelled CAT probe -> forms double strands where binding occurs
Treat hybridisation mix with Sl nuclease -> degrades all single stranded molecules
Electrophorese and autoradiograph
Only gene of interest remains
Medium Sensitivity
What is the principle behind the CAT assay?
Chloramphenicol + acetyl coenzyme A
Produces acetyl chloramphenicol
Acetyl chloramphenicol runs to a separate spot on gel than chloramphenicol
What is the principle behind the CAT assay?
Chloramphenicol + acetyl coenzyme A
Produces acetyl chloramphenicol
Acetyl chloramphenicol runs to a separate spot on gel than chloramphenicol
What are the main steps of the CAT assay
Chlorampheincol Acetyltransferase
Assay (CAT Assay)
Transfect construct
Incubate —48 hours
Isolate cytoplasmic extract
Incubate extract with 14C-Chloramphenicol and Acetyl
Coenzyme A
Extract Chloramphenicol products
Run Thin Layer Chromatography (TLC) to separate
14C- chloramphenicol and acetyl 14C-chloramphenicol
Autoradiograph
Why is the CAT assay considered a good method of assay
Visually very good at visualising levels of expression
Can clearly see between mono and diacetylated Chloramphenicol
If there is really high expression you might even see a triacetylate
- dont do this assay anymore though
What kind of CAT assay do we run now?
CAT sandwich ELISA
Use a plate coated with anti-CAT antibody
Blue colour
ELISA plate reader used
Nice simple assay which is also quantitative and results expressed graphically
Why do we often use the luciferase assay vector?
One of the quickest reporter assays
Luciferase RNA or enzyme product can be detected
Tube will immediately glow
Use a luminometer to read level of lfuorescence to determine amount f product
So much quicker and also quantitative
How does the b-galactosidase reporter assay work, why do we use it?
We use it because it is the cheapest method
It results in blue coloured cells which are read microscopically
It can be used as a stain -> cell/animals will be blue if they have been able to express the gene
How can B-galactosidase be used to prove expression of genes?
Used like a dye
Promoter connected to B-galactosidase
Expression of gene results in expression of blue dye
Promotive active in active genes
used in embryos - to display where certain genes are expressed etc
What is the TATAA box required for?
Transcription
The TATAA box is a DNA sequence essential for the initiation of transcription in eukaryotic cells.
What role does TATTAA play in the transcription process?
Acts in an orientational position
TATTAA helps position the transcription machinery correctly for effective transcription.
What happens to the catalytic subunit without TATAA?
It won’t bind correctly
The correct binding of the catalytic subunit is crucial for transcription initiation.
How does the positioning of the TATA box affect transcription?
Orientation and position dependent
The TATA box must be in a specific position relative to the gene start site to function properly.
What experimental setups were used to study the TATA box?
Reporter constructs with varied TATA box positions and orientations
These experiments included moving the TATA box closer to or further from the start site, placing it in incorrect orientations, or mutating it.
What was concluded from the alteration of the TATA box in experiments?
Interferes with transcription
Changes to the TATA box’s position or orientation negatively impact the transcription process.
Fill in the blank: The TATA box must be in a _______ position with respect to the start of the gene to function.
defined
A defined position is essential for the TATA box to effectively facilitate transcription.
What is the main focus of the experiment shown in Slide 59?
The effects of individual base mutations on transcription
The experiment involved generating 100 CAT reporter constructs with mutant promoters and analyzing RNA or CAT protein after transfection into cells.
What is the relationship between conserved sequences and transcription?
Mutations in conserved sequences tend to interfere with transcription
This indicates that conserved sequences play a critical role in the transcription process.
How many CAT reporter constructs were generated for the experiment?
100 CAT reporter constructs
Each construct had different mutant promoters.
What was observed when certain bases that bind NF1 were mutated?
A severe deficit in transcription
NF1 is a transcription factor, and its binding is crucial for transcription initiation.
What was the outcome of some mutations in certain regions regarding transcription?
Some mutations actually improved transcription
This highlights that not all mutations are detrimental; some may enhance transcriptional activity.
Fill in the blank: Mutations in _______ sequences have less of an effect on transcription.
non-conserved
Non-conserved sequences do not play as significant a role in transcription regulation.
True or False: All mutations in conserved sequences lead to improved transcription.
False
While some mutations may improve transcription, most tend to interfere with it.
What method was used to analyze the effects of the mutations on transcription?
Analysis of RNA or CAT protein
This analysis was performed after transfections into cultured cells.
What is absolutely required for transcription?
Promoter
The promoter region is essential for initiating the transcription process in molecular biology.
What is the function of the TATA box?
Acts as a core promoter in an orientation and position dependent manner
The TATA box is a crucial element in the promoter region that helps in the binding of RNA polymerase.
What happens to transcription if there are mutations in the TATA box?
Almost always completely abolishes transcription
Mutations can disrupt the binding of transcription factors and RNA polymerase.
What must be kept constant between the TATA box and the start site of transcription?
Sequence length
The specific sequence itself is unimportant, but the length must remain consistent for proper transcription initiation.
Which non-core promoter elements can be important for transcription?
CAAT, GC
These elements can enhance or regulate the transcription process, though they are not part of the core promoter.
True or False: The TATA box can be moved or flipped in orientation.
False
The TATA box’s position and orientation are critical for its function as a core promoter.
What is the primary function of metallothionein genes?
These respond to heavy metal ingestion
Metallothioneins are involved in the detoxification of heavy metals.
What key element is found in the promoter region of the metallothionein gene?
TATA box
The TATA box is essential for the initiation of transcription.
What type of region do metallothionein genes have in addition to the TATA box?
GC rich region
GC rich regions are often involved in the regulation of gene expression.
Which protein is indicated to bind to the GRE in the metallothionein promoter?
Steroid receptor
The steroid receptor is involved in the response to glucocorticoids.
What are the elements that contribute to metal induction in the metallothionein promoter?
MRE (Metal Response Elements)
MREs are specific sequences that facilitate the binding of metal-responsive proteins.
Fill in the blank: The regulatory region of a human metallothionein gene contains _______ elements in both its promoter and enhancer.
regulatory
Regulatory elements are crucial for gene expression control.
Which transcription factors are indicated to bind to the metallothionein enhancer?
AP2 and AP1
AP2 and AP1 are involved in the regulation of gene expression in response to various signals.
What is the role of the Methionine response sequence in the metallothionein gene?
Involved in response to methionine levels
Methionine response sequences help regulate the expression of genes based on methionine availability.
True or False: The metallothionein promoter contains elements for both metal induction and glucocorticoid response.
True
This dual responsiveness allows for fine-tuned regulation of metallothionein expression.
What does the presence of multiple binding sites in the metallothionein promoter indicate?
Regulatory complexity
Multiple binding sites allow for the integration of various signaling pathways.
What is the start site for transcription?
The start site is typically characterized by sequences such as Oct, GC, CAAT, TATAA, and INR
What are the components of a gene structure in the context of transcription?
The components include:
* Intron
* Exon 1
* Exon 2
* Intron 2
* Poly-adenylation signal
* Exon 3
What sequence do proteins bind to during transcription initiation?
Proteins bind to the TATA box sequence
How must proteins bind to the TATA box?
Proteins must bind in an orientation-specific manner
What does RNA polymerase II transcribe DNA into?
RNA polymerase II transcribes DNA into heterogeneous nuclear RNA (hnRNA)
True or False: RNA polymerase II can transcribe DNA from RNA.
False
Fill in the blank: A hypothesis is that a protein or a complex of proteins is binding to the _______.
TATA sequence
What is a logical hypothesis regarding RNA polymerase II and the TATA box?
RNA polymerase II is binding to the TATA box
What aspect of protein binding to the TATA box is crucial for transcription initiation?
Binding must be orientation and position dependent
What is the first step in proving the hypothesis that RNA polymerase II binds to the TATA box?
Designing an experiment to demonstrate the binding
What does RNA polymerase 1 transcribe?
rRNA
RNA polymerase 1 is essential for synthesizing ribosomal RNA, which is crucial for protein synthesis in cells.
What is the function of RNA polymerase 2?
Transcribes mRNA
RNA polymerase 2 plays a key role in synthesizing messenger RNA, which carries genetic information from DNA to ribosomes.
What does RNA polymerase 3 transcribe?
tRNA and small nuclear RNAs
RNA polymerase 3 is involved in the transcription of transfer RNA and small nuclear RNAs, important for protein synthesis and RNA processing.
What percentage of RNA in a cell is made up of rRNA?
16%
Ribosomal RNA is a significant component of the cellular RNA pool, playing a critical role in protein synthesis.
True or False: Regulation of rRNA transcription is as stringent as that of mRNA.
False
The regulation of rRNA transcription is not as strict as mRNA regulation, which is crucial for determining cell phenotype.
Fill in the blank: RNA polymerase 1 makes _______.
Ribosomal RNA
Ribosomal RNA is necessary in every cell for protein synthesis.
What is RNA polymerase II?
A multisubunit complex (~14 subunits) that transcribes DNA into hnRNA
hnRNA stands for heterogeneous nuclear RNA, which is the primary transcript in eukaryotic cells.
Can RNA polymerase II bind DNA independently?
No, it requires other factors (proteins) to facilitate DNA binding and transcription
This indicates that RNA polymerase II is not sufficient on its own for transcription.
What is the function of the L’ subunit of RNA polymerase II?
It has a carboxy terminal domain (CTD) repeat of 7 amino acids that can be phosphorylated
The CTD is critical for the regulation of transcription and processing of the RNA transcript.
What is the amino acid sequence of the CTD repeat in the L’ subunit?
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
This sequence is repeated many times and is involved in phosphorylation during transcription.
How many subunits does RNA polymerase II have?
Approximately 14 subunits
This multi-subunit structure contributes to its complex functionality in transcription.
Fill in the blank: RNA polymerase II transcribes DNA into _______.
hnRNA
hnRNA is the precursor to mRNA in eukaryotic cells.
True or False: The phosphorylation of the CTD repeats in RNA polymerase II is important for transcription.
True
Phosphorylation of the CTD is a key regulatory mechanism in the transcription process.
What happens if you mix RNA polymerase II with a sequence containing TATA?
It won’t bind without additional factors
The TATA box is a DNA sequence that indicates where a genetic sequence can be read and transcribed, but RNA polymerase II cannot bind to it alone.
What is the role of TFIID in transcription initiation?
TFIID is the first fraction identified that binds to DNA and helps in transcription initiation
TFIID is a complex that includes the TATA-binding protein (TBP) and is essential for the assembly of the transcription machinery.
Which transcription factor is necessary for RNA polymerase binding to TATA?
TFIIE
TFIIE is crucial for the initiation of transcription as it allows RNA polymerase to bind to the TATA box.
What is the function of TFIIA in the transcription process?
TFIIA aids in stabilizing the binding of TFIID to the TATA box but does not facilitate RNA polymerase binding on its own
TFIIA enhances the interaction between TFIID and DNA.
Which transcription factor is identified after TFIID and TFIIA that allows RNA polymerase to bind?
TFIIB
TFIIB is essential for the recruitment of RNA polymerase II to the promoter.
What does the TATAA box signify in a typical gene?
The TATAA box is a core promoter element that is crucial for transcription initiation
It is located approximately 25-30 base pairs upstream of the transcription start site.
Fill in the blank: The transcription initiation complex is built up of TFIID, TFIIA, TFIIB, and _______.
TFIIE
TFIIE is the final factor required for the initiation of transcription.
True or False: RNA polymerase binds to TATA without any additional transcription factors.
False
RNA polymerase requires the presence of transcription factors like TFIID, TFIIA, TFIIB, and TFIIE to bind to the TATA box.
What happens when RNA polymerase is added after TFIIB binds?
RNA polymerase binds, but transcription does not start until TFIIE is added
This indicates that TFIIE is necessary for the initiation of transcription.
What experimental approach was used to identify the transcription factors binding to DNA?
Making a nuclear extract, fractionating it, and testing fractions for DNA binding
This method allowed researchers to sequentially identify the transcription factors involved in the initiation complex.
What is the significance of the experimental finding that purified RNA alone does not bind to TATA?
It indicates that additional factors are required for RNA binding
This highlights the importance of transcription factors in the transcription process.
List the transcription factors involved in the transcription initiation complex in the order they were identified.
- TFIID
- TFIIA
- TFIIB
- TFIIE
These factors work sequentially to form the complete transcription initiation complex, allowing RNA polymerase to initiate transcription.
What is the role of TFII30 in molecular biology?
TFII30 is an inhibitor that causes non-functional binding.
What must be added for the transcription process to start?
TEIIA must be added.
What does TFIIB do in the transcription process?
TFIIB recruits RNA polymerase II and provides structure for it to work from.
List the order of transcription factors involved after TFIIB.
- TFIIF
- TFIIE
- TFIIH
True or False: TFIIB functions independently without other transcription factors.
False
Fill in the blank: TFIIB recruits _______.
[RNA polymerase II]
What is the significance of TEIIA in the transcription process?
TEIIA is necessary for the transcription process to initiate.
What is TFIID composed of?
TBP and approximately 10 TAFs
Total size is around 800kDa
What does TBP recognize and bind to?
TATA box sequence
In which groove of DNA does TBP bind?
Minor groove
What effect does TBP binding have on the DNA strand?
Results in the bending of the DNA strand
What can inhibit TBP binding?
TF_230
True or False: TBP binds to the major groove of DNA.
False
What is the role of TFIIA in relation to TBP?
May bind DNA in conjunction with TBP
What is the size of TBP?
Approximately 30kDa
What is the total size of TFIID?
Approximately 800kDa
Fill in the blank: TBP binds to the _____ groove of DNA.
minor
What is suggested about TFIIA’s relationship with TFIIB?
It is quite possible that TFIIA is part of TFIIB
What is the significance of TBP binding to the minor groove?
It probably helps the transcription process
What is the role of TFIIA in transcription?
Binds to TFIID, possibly in solution and may activate TBP by relieving the repression caused by TFTF
TFIIA binds upstream of TFIID to facilitate transcription initiation.
What does TFIIB interact with?
Interacts with TFID and TFIIF
TFIIB binds upstream of TBP to a BRE sequence in some promoters.
Where does TFIIB bind in relation to TBP?
Binds upstream of TBP to a BRE sequence and downstream to the minor groove of DNA
This positioning may help align the transcription complex correctly.
What is the main role of TFIIB?
To recruit RNA polymerase II
TFIIB acts as a bridge between TFIID and RNA polymerase II.
How does TFIIB contribute to the transcription process?
Provides a surface (domain) that is recognized by RNA polymerase II
This interaction is crucial for the assembly of the transcription complex.
True or False: TFIIB can allow transcription to occur on its own.
False
TFIIB does not initiate transcription by itself; it requires other factors.
TFIIB is known to interact with which other transcription factors?
D and F
These interactions help in aligning the transcription process.
Fill in the blank: TFIIB binds upstream from TFIID from the start site of transcription and probably aligns the whole _______.
transcription process
This alignment is essential for proper transcription initiation.
What is the function of TFIIB in relation to the transcription complex?
Acts as a bridge between TFIID and RNA polymerase II
This bridging is crucial for the assembly and stability of the transcription complex.
What is the composition of TFIIF?
Composed of 4 subunits
Includes 2 X RAP 74 and 2 X RAP 30
What is one of the functions of TFIIF?
Interacts with CTD phosphatase
CTD refers to the C-terminal domain of RNA polymerase II
How does TFIIF interact with DNA?
Interacts with DNA on either side of the TATA box
TATA box is a core promoter element
What role does TFIIF play in relation to RNA Polymerase I?
May bind to and bring RNA Polymerase I to the transcription complex
What complex does TFIIF stabilize?
Stabilises TBP - DNA – TFIIB complex
What is a requirement for elongation in transcription?
Required for elongation
Which transcription factors does TFIIF facilitate binding with?
Required for TFIIE and TFIIH binding
What is the composition of TFIIE?
Heterotetramer (2 X 34kDa, 2 X 56kDa)
To which molecule does TFIIE bind directly?
Binds directly to RNA polymerase II
How does TFIIE interact with DNA?
Binds to DNA nonspecifically
What is one of the roles of TFIIE in transcription?
Mediates assembly of TFIIH
What possible activity does TFIIE have?
Possible helicase activity
What does TFIIE do to the helicase activity of TFIIH?
Represses helicase activity of TFIIH
What happens to TFIIE upon phosphorylation of the CTD?
Phosphorylation of CTD releases TFIIE and allows unwinding of DNA
Fill in the blank: TFIIF is involved in bringing _______ to the transcription complex.
[RNA Polymerase I]
TFIIF stays on during which phase of transcription?
Elongation phase
What is the major role of TFIIF?
Recruit H
True or False: TFIIF definitely has helicase activity.
False
What is the effect of CTD tail phosphorylation on TFIIE?
Releases TFIIE
What is the primary function of TFIIH in molecular biology?
TFIIH has DNA repair activity and phosphorylates the CTD tail of RNA polymerase II.
TFIIH is a multi-subunit complex involved in transcription and nucleotide excision repair.
What activities are associated with TFIIH?
TFIIH is associated with the following activities:
* ATPase activity
* Helicase activity
* Kinase activity
These activities are crucial for its role in transcription and DNA repair.
What does kinase activity refer to?
Kinase activity refers to phosphorylation activity.
Kinases are enzymes that catalyze the transfer of phosphate groups.
What happens to the CTD tail of RNA polymerase II upon phosphorylation?
Phosphorylation leads to the release of the unphosphorylated CTD tail from TBP and initiates transcription elongation.
TBP stands for TATA-binding protein, which is part of the transcription initiation complex.
Fill in the blank: The unphosphorylated CTD tail of RNA polymerase II is bound to _______.
[TATA box protein]
True or False: Phosphorylation of the CTD tail of RNA polymerase II is necessary for transcription to occur.
True
Phosphorylation of the CTD tail is a critical step in the transition from transcription initiation to elongation.
How does the phosphorylation of the CTD tail affect transcription?
It leads to transcription elongation after release from TBP.
This process is essential for the synthesis of RNA from the DNA template.
What is the state of RNA polymerase II when the initiation complex is built up?
Unphosphorylated
The unphosphorylated state of RNA polymerase II is essential for the formation of the initiation complex.
Which transcription factor phosphorylates RNA polymerase II as a requirement for the elongation stage?
TFIIH
TFIIH is known for its role in phosphorylation which is crucial for transitioning into the elongation phase of transcription.
What is one result of the phosphorylation of RNA polymerase II?
Release of many transcription factors
Phosphorylation allows RNA polymerase II to transition into the elongation phase by releasing transcription factors that are no longer needed.
What enzyme does RNA polymerase II bind after phosphorylation?
Capping enzyme (guanylyl transferase)
The capping enzyme is involved in adding a protective cap to the 5’ end of the RNA transcript.
What type of factors does RNA polymerase II bind to after phosphorylation?
Splicing factors
Splicing factors are essential for the RNA splicing process, which removes introns from the pre-mRNA.
What apparatus does RNA polymerase II bind to after being phosphorylated?
Cleavage/polyadenylation apparatus
This apparatus is crucial for the processing of pre-mRNA by cleaving and adding a poly-A tail.
What is the composition of the Mediator Complex?
At least 20 subunits
The Mediator Complex plays a crucial role in transcription regulation.
Is the Mediator Complex required for basal transcription?
Not required
Basal transcription can occur without the Mediator Complex.
What types of signals does the Mediator Complex transduce?
Positive and negative signals
These signals come from gene-specific activators and repressors.
Can the Mediator Complex bind to DNA?
Cannot bind DNA
This characteristic differentiates it from some other transcription factors.
What activity does the Mediator Complex regulate?
TFIIH CTD kinase activity
This regulation is important for the phosphorylation process during transcription initiation.
What is the role of the promoter in transcription?
Sufficient for transcription at basal levels
The promoter is necessary for initiating transcription.
How does the promoter act in relation to transcription?
In an orientation and position dependent manner
This means the promoter’s effectiveness can vary based on its location and orientation.
What does the promoter bind to?
Components of transcription initiation complex
This binding is essential for the initiation of transcription.
What factors may influence the promoter’s activity?
Factors bound at other DNA sites
These factors can enhance or repress the transcription process.
What is one function of enhancers in gene expression?
Upregulate and downregulate transcription
Enhancers respond to extracellular activities and environmental signals.
Fill in the blank: The Mediator Complex is required for _______.
[transcription regulation]
While not required for basal transcription, it is essential for regulated transcription.
True or False: The Mediator Complex is essential for all types of transcription.
False
It is not necessary for basal transcription but is crucial for regulated transcription.
What may the Mediator Complex bind to in relation to tissue-specific gene expression?
Some factors required for tissue-specific gene expression
This allows certain genes to be active only in specific tissues.
What are enhancers in molecular biology?
Sequences not absolutely required for transcription that modulate the rate of transcription from a promoter.
How do enhancers affect transcription levels?
They can increase or decrease transcription levels.
What is the role of the glucocorticoid response element?
Involves higher levels of transcription.
What is the effect of including additional bases around a promoter?
It results in more transcription.
What is the S1 nuclease protection assay used for?
To study the components of typical transcriptional regulation.
What is the relationship between transcription factors and transcription levels?
Transcription factors influence transcription through mechanisms like phosphorylation.
What happens to histones during transcriptional activation?
Histones are acetylated.
Fill in the blank: Enhancers are located in the _______ regions of genes.
flanking
True or False: Transcription factors typically have multiple domains, including phosphorylation sites.
True
What is the expected structure of transcription factors?
Dimerized with various domains including phosphorylation sites and dimerization groups.
What is the significance of mediator in transcription?
It influences transcription factors like TFIIH and promotes phosphorylation.
What are the components of typical enhancer binding proteins?
• DNA binding domain
• Dimerisation domain
• Transcriptional activation domain
• Ligand binding domain
• Phosphorylation / modification site
These components are crucial for the function of transcription factors in gene regulation.
What is the function of the DNA binding domain in transcription factors?
It allows the transcription factor to bind to specific DNA sequences.
The DNA binding domain is essential for recognizing and attaching to the promoter or enhancer regions of genes.
What is the role of the dimerisation domain in transcription factors?
Facilitates protein-protein interactions necessary for dimer formation.
Dimerization is important for the function of many transcription factors, allowing them to work together.
What is the purpose of the transcriptional activation domain?
It mediates interactions with other proteins to activate transcription.
This domain enhances the transcriptional activity of RNA polymerase and other co-activators.
What does the ligand binding domain do in transcription factors?
Binds to small molecules or ligands that can influence transcriptional activity.
The binding of ligands can lead to conformational changes that activate or repress transcription.
What is the significance of the phosphorylation/modification site?
It allows for post-translational modifications that regulate transcription factor activity.
Phosphorylation can alter the stability, localization, or activity of transcription factors.
What is the role of TAFs in transcription?
They are part of the transcription pre-initiation complex that assists RNA polymerase binding.
TAFs are important for recognizing promoter elements and facilitating the assembly of the transcription machinery.
Which transcription factor is represented by TBP?
TATA-binding protein
TBP is a component of the transcription factor IID complex and is essential for the initiation of transcription.
What does TFIIH do in the transcription process?
It is involved in the unwinding of DNA and phosphorylation of the RNA polymerase II C-terminal domain.
TFIIH has helicase activity, which is crucial for opening the DNA double helix during transcription initiation.
What is the role of Mediator in transcription?
It serves as a bridge between transcription factors and RNA polymerase II.
Mediator helps integrate signals from various transcription factors to regulate gene expression.
Fill in the blank: The DNA must be in an _______ format for transcription to occur.
open uncondensed
Open chromatin allows transcription factors and RNA polymerase access to the DNA.
True or False: Transcription factors can influence transcription from locations that are 50,000 base pairs away.
True
This demonstrates the ability of enhancers to regulate transcription over long distances.
What is the function of the polymerase along with the CDL tail?
It is recruited by TFIIH during transcription initiation.
The C-terminal domain (CDL) of RNA polymerase II is critical for its interaction with transcription factors.
What are tissue-specific genes?
Genes that are expressed in only a single cell type
Examples include Growth Hormone in somatotroph cells and Prolactin in mammotroph cells of the pituitary gland.
Which cell types express Growth Hormone?
Somatotroph cells in the anterior pituitary
Growth Hormone is present in every cell but is only active in somatotroph cells.
Which cell types express Prolactin?
Mammotroph cells in the anterior pituitary
Prolactin is another example of a tissue-specific gene.
How is gene transcription activated in specific cell types?
Factors only present in these cell types are required to activate the transcription of these genes
These factors often bind to specific DNA sequences in the gene’s promoter.
True or False: All genes are expressed in every cell type of the body.
False
Many genes are only active in specific cell types, such as somatotrophs and mammotrophs.
What is the role of DNA sequences in gene activation?
They are bound by proteins that are only found in specific cell types
This binding is crucial for the activation of tissue-specific genes.
Fill in the blank: The likely way tissue-specific gene expression occurs is through a _______ in the promoter of these genes being bound by specific proteins.
DNA sequence
This process is essential for differentiating pluripotent stem cells into specific cell types.
What is the significance of pluripotent stem cells in differentiation?
They can develop into various cell types, such as neurons or liver cells
Differentiation is triggered by specific signals during development.
What is the function of activators in transcription?
They influence the transcription process to occur
Activators mediate the binding of RNA polymerase and other transcription factors.
What are co-activators?
Proteins that assist activators in enhancing gene transcription
They do not bind to DNA directly but interact with other transcription factors.
What are co-repressors?
Proteins that inhibit gene transcription
They bind to transcription factors and prevent the transcription machinery from assembling.
What role do elongation factors play in transcription?
They assist RNA polymerase during the elongation phase of transcription
Elongation factors help in the process of synthesizing RNA from the DNA template.
What are splicing factors?
Proteins involved in the splicing of pre-mRNA
They play a crucial role in the modification of RNA after transcription.
What is the function of processing factors in RNA transcription?
They modify RNA transcripts after they are synthesized
This includes capping, polyadenylation, and splicing.
What is RNA polymerase II responsible for?
Transcribing mRNA from DNA
It is essential for the synthesis of protein-coding genes.
What is the function of the 5’ flanking region in the growth hormone gene?
It contains transcription factor binding sites for gene regulation.
This region is critical for the initiation of transcription by recruiting necessary proteins.
Which transcription factor is specifically mentioned as binding to the GH promoter?
Pit-1
Pit-1 plays a crucial role in the expression of growth hormone.
What is the consensus sequence for the binding elements in the growth hormone gene?
AAATATNCAT
This sequence is important for the binding of transcription factors to the promoter region.
What are GRE-like elements associated with?
Glucocorticoid receptor binding
These elements are involved in the regulation of genes by glucocorticoids.
Fill in the blank: The transcription factor _______ is essential for growth hormone gene expression.
Pit-1
True or False: The growth hormone promoter is only influenced by proteins from the pituitary gland.
False
While pituitary extracts are mentioned, other factors may also influence the promoter.
What is the significance of the TATAA sequence in gene promoters?
It serves as a core promoter element for transcription initiation.
The TATAA box is a common feature in many eukaryotic promoters.
What does the term ‘unbound’ refer to in the context of transcription factors?
Transcription factors that are not currently attached to DNA sequences.
Unbound transcription factors can become active when conditions change.
What role do consensus sequences play in gene regulation?
They provide specific binding sites for transcription factors.
This specificity is crucial for the precise regulation of gene expression.
How does Pit-1 interact with the growth hormone promoter?
By binding to specific elements in the promoter region.
This interaction is vital for activating the transcription of the growth hormone gene.
What is the role of transcription in molecular biology?
Transcription is the process of copying a segment of DNA into RNA.
What type of cell is specifically mentioned in the context of transcription?
Pituitary cell.
What is the result of transfecting a liver cell in this context?
No transcription occurs.
What is involved in the process of transcription factor purification?
Isolating specific proteins that regulate transcription.
What are the steps involved in gene cloning mentioned?
Sequence protein and clone gene.
Fill in the blank: Transcription occurs in _______ cells.
[pituitary]
True or False: Transcription occurs in liver cells after transfection.
False.
What process is used to introduce foreign DNA into a cell?
Transfection.
What is the purpose of fraction collection in molecular biology?
To isolate and analyze specific components from a mixture.
What is the role of miRNA in translation inhibition?
miRNA inhibits translation of target RNA
miRNA can bind imperfectly to multiple mRNAs.
What is the function of siRNA?
siRNA cleaves target RNA
siRNA is involved in the RNA interference pathway.
What does the acronym PIT1 stand for?
PIT1 is a transcription factor from pituitary cells
It regulates gene expression in various tissues.
True or False: A single miRNA can act on multiple mRNAs.
True
This is due to the imperfect binding of miRNA.
Fill in the blank: miRNA can act on multiple _______.
mRNAs
This is a result of its imperfect binding ability.
What happens to the expression of miRNA in cancers?
Cancers have dysregulation of miRNA
This can lead to altered gene expression and cancer progression.
What is the significance of imperfect binding in miRNA function?
Imperfect binding allows a single miRNA to regulate multiple target RNAs
This increases the regulatory potential of miRNAs.
Where does the PIT1 transcription factor originate?
PIT1 originates from pituitary gland cells
It is crucial for the development of certain hormones.
What is the process of transfecting a gene of interest into a liver cell?
Transfection introduces foreign DNA into cells
This can be used to study gene function in non-native cells.
What is the primary difference between miRNA and siRNA?
miRNA inhibits translation, while siRNA cleaves RNA
Both are involved in gene regulation but act through different mechanisms.
What is the target of miR-196a in humans?
HOXB8 mRNA
miR-196a regulates HOXB8, which is involved in developmental processes.
What is the target of miR-166 in plants?
PHAVOLUTA mRNA
miR-166 is important for plant development and leaf patterning.
What is the overall impact of miRNA on gene expression?
miRNA can downregulate gene expression
This regulation can impact various cellular processes and pathways.
What are miRNAs?
Short non-coding RNA molecules that regulate gene expression
miRNAs are crucial in post-transcriptional regulation.
Approximately how many miRNA genes are believed to exist in the human genome?
~1000 genes
These miRNAs are involved in regulating a significant portion of gene expression.
What proportion of human genes are regulated by miRNAs?
~1/3 of all human genes
This indicates the extensive role of miRNAs in gene regulation.
How is miRNA made?
Through a process involving self-complementarity and the dicer complex
This process results in a double-stranded hairpin structure that is processed further.
What is the role of the dicer complex in miRNA processing?
Removes the hairpin structure from the precursor miRNA
Dicer is essential for producing functional miRNA.
What complex binds to the processed miRNA after dicer removes the hairpin?
RISC complex
RISC stands for RNA-induced silencing complex, which plays a crucial role in gene regulation.
What is the major protein involved in the RISC complex?
Argonaute protein
Argonaute is integral to the functionality of RISC in gene silencing.
What happens to the miRNA strand after it is processed by dicer?
Left with a single strand
This single strand is then ready to interact with target mRNAs.
What are the two possible outcomes when a single strand of miRNA binds to a complementary strand?
- Perfectly bound = degradation of strand
- Imperfectly bound = failure to translate
These outcomes determine the effectiveness of gene silencing.
Fill in the blank: The process of miRNA binding can lead to either degradation of the target strand or _______.
failure to translate
This highlights the role of miRNAs in regulating gene expression.
