Lecture 9 - 10 Flashcards
What is the nucleoli?
the site of rRNA and ribosome synthesis.
What is the nucleoplasm
the fluid inside of the nucleus where solutes are dissolved.
What is the nuclear matrix?
a protein fiber network.
What is the nuclear envelope?
Two membranes separated by nuclear space. the two membranes are fused together via the nuclear pore.
what is the nuclear lamin?
the inner surface of the nuclear envelope. made of intermediate filaments (lamin is actually a type of cytoskeleton protein).
What is the nuclear localization signal?
a positively charged amino acid sequence found within a protein.
when the protein nears the nuclear pore complex, importin will read the signal and bind to the protein.
-it is also categorized as a type of consensus sequence
What is importin? What is its function?
recognizes the NLS sequence. made of two subuntis. beta subunit binds to NLS. alpha subunit binds to FG proteins.
What is exportin?
a protein that reads the nuclear export signal. it transports large molecules across the nuclear membrane.
Describe the physical shape of the nuclear pore complex.
- They are 30x larger than a ribosome.
- they have octagonal symmetry
- FG proteins (Phenylalaline and Glycine) face the cytoplasmic side (move like wiggly worms)
- opening of the pore is lined with proteins
What are FG proteins? Describe their structure.
They are repeats of Phenylalaline and Gylcine.
they are very disordered (no secondary structure), which is what gives them their wiggly worm movements
-they are hydrophobic because of Phenylalanine.
-found on the cytoplasmic side of the nuclear pore.
What is diffusion?
the movement of a particle from an area of high concentration to an area of low concentration.
Energy is required to maintain or create a concentration gradient
Briefly describe what GEF is.
Guanine Nucleotide Exchange Factor, an exchange factor that activates a G-protein. exchanges GDP to GTP.
example: RCC1
Briefly describe GAP.
GTPase Activating Protein. Turns off G-proteins by hydrolyzing GTP - GDP.
Example: Ran-GAP1
Briefly describe what Ran is.
a type of g-protein that is not membrane bound. it is turned on by GEF (RCC1), and turned off by Ran-GAP1 (GAP). It binds to the beta-subunit of the importin complex.
What is RCC1?
a type of GEF. it turns on Ran g-protein. Ran-GDP -> Ran-GTP. found in high concentrations within the nucleus.
What is RanGAP1?
a type of GAP. it turns off Ran g-protein. Ran-GTP ->Ran-GDP. found in high concentrations within the cytoplasm
Briefly describe importin.
a protein made of an alpha and beta subunit. The beta subunit recognizes the NLS amino acid sequence. the alpha-subunit binds to FG proteins of the nuclear pore.
Briefly describe spatial expression.
cells differentiate depending where they are travelling to in the body (travel regulated by hormones, TFs, etc.)
What is temporal Expression?
depending on the age of the body/cells (fetus, child, young adult, adult, senior) different hormones and TF will be active.
What are expression patterns?
different combinations of TFs and hormones presented at different locations and time in the body.
What are some of the general patterns of expression?
- Housekeeping genes (genes that encode basic cell functions)
example: ATPsynthase, Tubulin, RNA/DNA polymerase - Genes expressed while stem cells differentiate, then are turned off (Temporal)
- Genes only expressed in specialized cells (spatial)
- genes express in response to certain stimuli (hormones, environmental effects/epigenetics).
What is differential gene expression?
each cell type only expresses its subset of genes (those that are transcriptionally active in the cell).
What are the five levels of gene regulation?
- Epigenetic Level
- Transcription Level
- mRNA processing
- Translational
- Post-Translation
What is a transcriptional activator? Where is it found?
Transcription Factors that promote the transcription of a gene. found inside the promoter region.
What is a transcriptional repressor? where is it found?
Transcription Factors that inhibit transcription. also found in the promoter region of a DNA strand.
Describe the general overall structure of Transcription Factors.
- TFs have domains that interact with DNA
- most TFs operate as dimers (hetero or homodimers). they bind together at dimerization domains.
example: DNA binding domain, Regulatory Domain
Describe a DNA binding domain.
TFs fit into the major groove of DNA. The TFs amino acids bind to the DNAs nitrogenous bases via Hydrogen Bonding.
Describe a Regulatory Domain on a TF.
when TF are bound to DNA, the regulatory domain is open for other molecules to influence transcription.
Example of other molecules: proteins/TFs/components of the initiation complex/co-activator proteins/etc.
Name the common Transcription Factor binding motifs.
Zinc-Finger Motif
Helix-Loop-Helix Motif
Leucine-Zipper Motif.
Describe the Zinc-Finger motif.
multi “finger-shaped” projections of protein that fit into the major grooves of DNA.
Describe the Helix-Loop-Helix motif.
forms homo and heterodimers.
Describe the Leucine-Zipper motif.
two helices “zipped” together to form a coild-coil structure as dimers.
What are the requirements for a Transcription Factor to be active?
- must be produced by transcription
- must be in the nucleus (import is regulated)
- often activated by post-translation modifications (phosphorylation)
Describe the characteristics of the core promoter.
- defines the position of the start site (1-40bp away from the gene)
- TATA box is located within (note, not all genes have a TATA box associated with them)
- TFs assemble the pre-initiation complex at this site
Name some of the molecules that would be associated with the core promoter.
- TATA binding protein
- TBP-associated factors
- RNA-POL II
- pre-initiation complex
- TFIID (recognizes TATA sequence)
- TFIIH (acts as a protein kinase-phosphorylates)
Do all core promoters have a TATA box?
No.
Describe the proximal promoter.
- found -40 to -150 bp away from TATA box (-1)
- contains CAAT and GC boxes
- the quantity and type of proximal and distal elements regulate the frequency of transcription
How can the proximal promoter be modified?
When the “C”s within the GC box are methylated, the gene becomes transcriptionally inactive. When C’s become methylated TF’s are unable to bind to the methylated nucleotide.
What molecules are associated with the proximal promoter?
Transcription Factors NF1 and SP1.
What is the function of NF1?
Nuclear Factor 1 recruits a co-activator required for RNA Polymerase to function
Describe the distal promoter.
- found -500 to -1000bp away from TATAbox (-1)
- contains response elements.
What are response elements? Give an example.
Response elements are specific transcription factors related to one gene or a group of related genes. They can inhibit or activate transcription of a gene.
Example: PEPCK gene ( phosphoenolpyruvate carboxykinase - an enzyme required to make glucose)
PEPCK is expressed in the liver. The gene becomes active (is transcribed/translated) when the body is hungry or under stress.
Define enhancer regions.
- DNA sequences found within 10,000bp of a gene
- they can be located before/after or even inside the target gene
- they are similar to distal promoters
What is a clamp protein?
a protein that binds chromosomes to proteins of the nuclear matrix.
Briefly describe how the different promoter types modulate transcription.
- TFs bind to response elements in the distal promotor.
- Enhancer-bound activators fold around to interact with the promotor region. (this is similar to how mRNA folds into a U-shape when it is being transcribed
- Together Response Elements and Enhancers recruit Co-activators
- Co-activators enhance RNA-POL activity
- all binding and recruiting is controlled by a multi-protein complex called mediator.
How do co-activators enhance RNA-POL activity?
- assist in the assembly of the pre-initiation complex
- alter the chromatin state by changing the histones so DNA is more accessible for transcription
what is CBP and what is its function?
-CREB-binding Protein, is a histone acetyltransferase that acts as a co-activator.
it addes an acetyl group to the histones, causing DNA to loosen around the histones
Where specifically does CBP operate?
CREB-binding Protein targets histones located in the proximal and core promoters.
Explain the process of a functional Glucocorticoid Response Element.
- Glucocorticoid receptor binds to distal Glucocorticoid Response Element and recruits CREB-binding Protein
- CBP adds an acetyl group to the histone and causes DNA to loosen its association with those histones
- acetylated histones recruit SW1/SNF, which in turn remove the histones in the core promoter region. Making DNA more accessible
- when the core promoter is exposed, the pre-initiation complex can assemble.
briefly explain what SW1/SNF is and what its function is.
Switch/Sucrose Nonfermenting, these molecule is an ATP dependent remodeling complex.
These molecules remove the histone in the core promoter region.
what does TFIID do?
TFIID also uses histone acetyltransferase activity at the core promotor region.
How would you figure out the function of a promoter? Explain the process
Use Deletion Mapping.
- fuse a promotor into 5’ coding sequence of a reporter gene such as GFP
- perform deletions within different parts of the promoter
- introduce these mutations into a cell (one mutation at a time) and measure how much GFP is being expressed relative to the full-length promoter
- if fluorescence is low (gene expression) is low, you have deleted an important piece of the promoter
What would happen if you deleted the TATA box from the core promoter?
you would get very little gene expression. only 38% of gene would be expressed.
what would happen if you deleted base pairs -40 to -70 bp away from the +1?
gene expression would still be relatively high, about 95%.
What would happen if you deleted the GC box in the proximal promoter?
you could get very little gene expression. only about 14%
what would happen if you deleted the CAAT box in the proximal promoter?
only 50% of the gene would be expressed.
what if you deleted a repression element?
the gene would be transcribed more. about 114%
Why is GFP such a handy tool?
when you attached a GFP to the promoter region of a gene, that gene will fluoresce when it is transcribed.
The nice thing is, you can see where in the cell/body the gene is being expressed, when the gene is being expressed and at what frequency.
What does potency mean in reference to a cell?
A cell’s ability to differentiate into other cells.
Define totipotency.
A cell’s ability to differentiate into any of our adult cell types.
Name the different stages and potency a cell will undergo.
-embryonic stem cells -> differentiate into progenitor cell types (they are pluripotent) –> three germ layers endoderm, mesoderm, ectoderm –>adult stem cells (multipotent)
Give examples of adult stem cells.
intestinal cells (only last a few days), blood cells (last days to months depending on type) , neurons (it takes years to grow another)
What are adult stem cells capable of?
- self renewal (produces more cells of the same type)
- differentiation into two or more mature cell types
- daughter cells can only divide ~60 times (telomere shortening)
- most, if not all, organs contain stems cells capable of replacing the different tissues in the organ
What is a master switch?
A transcription factor that controls many many other transcription factors.
How do researchers manipulate master switches? Give an example.
Master switches are used to induce differentiation in a stem cell.
Example: a cell expressing MyoD transcription factor will cause a fibroblast cell (connective tissue) to turn into a muscle cell.
We can also grow eyes on a leg by altering the master switch in a cell.
Explain the general process of what happens when a stem cell divides.
- one of the daughter cells will continue to divide, the other will remain as a stem cell
- during subsequent divisions, the cells receive signals (hormones) that activate transcription factors
- the different combination of transcription factors influence down stream TFs, guiding the cell into specific cell types
Define cell memory.
A master switch will remain on in differentiated cells to maintain that state.
How does a differentiated cell remain in its final state?
A phenomenon known as cell memory. A master switch will remain on because they bind to their own promoter region. Therefore they are continually transcribed (positive feedback loop)
Do stem cells require cell memory as well?
Yes. Master switches maintain stem cells, and can also influence other stem cell states.
What state is the DNA in when located inside a totipotent cell?
DNA mostly in a euchromatin state.
Except for Constitutive Heterochromatin
How is the state of DNA effected by the differentiation of a cell?
DNA/genes involved in the process of differentiation, or in specialized cells will remain in a euchromatin state.
DNA/Genes not involved in differentiation, or not required for the specialized cells functioning will become condensed into heterochromatin.
Describe the theory/application of stem cell therapy.
- The idea is to inject the desired genes into a stem cell
- induce cellular replication in the cell
- then introducing master switches and other TFs, differentiate the cell into whichever the patient is lacking.
Where do researchers harvest stem cells from?
- Totipotent embryonic stem cells (create an embryo just to remove the stem cells from it. slightly unethical)
- Induce Pluripotent stem cells
What is iPS? How does it work?
Induced Pluripotent Stem cells.
A differentiated cell is removed from a patient. Master switches (OCT4, SOX2, KLF4) and desired genes are introduced into the cell, causing the cell to somewhat de-differentiate and express the genes we have inserted.
Cell replicates and those cells are introduced back into the patient.
Generally speaking, what is transcriptional repression? Give an example.
Genes can be “turned off” by repressor TFs binding to the response elements associated with genes.
Example: Histone Deacetylases and Methyltransferases
What is HDAC?
Histone DeAcetlyases Complex.
These remove acetyl groups from histones, causing DNA to wind more tightly around the histone. Silences gene activity
What is Histone Methyltransferases? How does it work?
an enzyme that adds methyl groups to the 5’ carbon of cytosine (ALWAYS 5’).
The methyl group acts as a tag, recruiting inhibitory proteins to the 5-methylcytosine bases so other TFs cannot bind to the DNA.
Is methylation reversible?
It is. however, methylated DNA often remains in that state during cell division.
What is Maintenance Methyltransferase?
An enzyme that maintains the methylated state of the 5-methylcytosine bases in newly transcribed mRNA.
How is methylated DNA maintained during cell division?
Maintenance Methyltransferase will travel along the newly transcribed mRNA and methylate the same cytosines that were methylated in the template strand.
What types of DNA modifications remain in newly synthesized DNA?
Methylation and Histone Tail Modifications
How do we know DNA methylation can be passed on to the next generation?
By using Illumina sequencing we can find where DNA methylation has occurred throughout the genome.
