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Bio 2520 > Lecture 9 - 10 > Flashcards

Lecture 9 - 10 Flashcards

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Biology 101 flashcards
1
Q

What is the nucleoli?

A

the site of rRNA and ribosome synthesis.

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2
Q

What is the nucleoplasm

A

the fluid inside of the nucleus where solutes are dissolved.

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3
Q

What is the nuclear matrix?

A

a protein fiber network.

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4
Q

What is the nuclear envelope?

A

Two membranes separated by nuclear space. the two membranes are fused together via the nuclear pore.

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5
Q

what is the nuclear lamin?

A

the inner surface of the nuclear envelope. made of intermediate filaments (lamin is actually a type of cytoskeleton protein).

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6
Q

What is the nuclear localization signal?

A

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

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7
Q

What is importin? What is its function?

A

recognizes the NLS sequence. made of two subuntis. beta subunit binds to NLS. alpha subunit binds to FG proteins.

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8
Q

What is exportin?

A

a protein that reads the nuclear export signal. it transports large molecules across the nuclear membrane.

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9
Q

Describe the physical shape of the nuclear pore complex.

A
  • 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
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10
Q

What are FG proteins? Describe their structure.

A

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.

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11
Q

What is diffusion?

A

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

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12
Q

Briefly describe what GEF is.

A

Guanine Nucleotide Exchange Factor, an exchange factor that activates a G-protein. exchanges GDP to GTP.
example: RCC1

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13
Q

Briefly describe GAP.

A

GTPase Activating Protein. Turns off G-proteins by hydrolyzing GTP - GDP.
Example: Ran-GAP1

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14
Q

Briefly describe what Ran is.

A

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.

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15
Q

What is RCC1?

A

a type of GEF. it turns on Ran g-protein. Ran-GDP -> Ran-GTP. found in high concentrations within the nucleus.

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16
Q

What is RanGAP1?

A

a type of GAP. it turns off Ran g-protein. Ran-GTP ->Ran-GDP. found in high concentrations within the cytoplasm

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17
Q

Briefly describe importin.

A

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.

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18
Q

Briefly describe spatial expression.

A

cells differentiate depending where they are travelling to in the body (travel regulated by hormones, TFs, etc.)

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19
Q

What is temporal Expression?

A

depending on the age of the body/cells (fetus, child, young adult, adult, senior) different hormones and TF will be active.

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20
Q

What are expression patterns?

A

different combinations of TFs and hormones presented at different locations and time in the body.

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21
Q

What are some of the general patterns of expression?

A
  • 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).
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22
Q

What is differential gene expression?

A

each cell type only expresses its subset of genes (those that are transcriptionally active in the cell).

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23
Q

What are the five levels of gene regulation?

A
  1. Epigenetic Level
  2. Transcription Level
  3. mRNA processing
  4. Translational
  5. Post-Translation
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24
Q

What is a transcriptional activator? Where is it found?

A

Transcription Factors that promote the transcription of a gene. found inside the promoter region.

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25
Q

What is a transcriptional repressor? where is it found?

A

Transcription Factors that inhibit transcription. also found in the promoter region of a DNA strand.

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26
Q

Describe the general overall structure of Transcription Factors.

A
  • 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
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27
Q

Describe a DNA binding domain.

A

TFs fit into the major groove of DNA. The TFs amino acids bind to the DNAs nitrogenous bases via Hydrogen Bonding.

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28
Q

Describe a Regulatory Domain on a TF.

A

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.

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29
Q

Name the common Transcription Factor binding motifs.

A

Zinc-Finger Motif
Helix-Loop-Helix Motif
Leucine-Zipper Motif.

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30
Q

Describe the Zinc-Finger motif.

A

multi “finger-shaped” projections of protein that fit into the major grooves of DNA.

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31
Q

Describe the Helix-Loop-Helix motif.

A

forms homo and heterodimers.

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32
Q

Describe the Leucine-Zipper motif.

A

two helices “zipped” together to form a coild-coil structure as dimers.

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33
Q

What are the requirements for a Transcription Factor to be active?

A
  • must be produced by transcription
  • must be in the nucleus (import is regulated)
  • often activated by post-translation modifications (phosphorylation)
34
Q

Describe the characteristics of the core promoter.

A
  • 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
35
Q

Name some of the molecules that would be associated with the core promoter.

A
  • TATA binding protein
  • TBP-associated factors
  • RNA-POL II
  • pre-initiation complex
  • TFIID (recognizes TATA sequence)
  • TFIIH (acts as a protein kinase-phosphorylates)
36
Q

Do all core promoters have a TATA box?

A

No.

37
Q

Describe the proximal promoter.

A
  • 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
38
Q

How can the proximal promoter be modified?

A

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.

39
Q

What molecules are associated with the proximal promoter?

A

Transcription Factors NF1 and SP1.

40
Q

What is the function of NF1?

A

Nuclear Factor 1 recruits a co-activator required for RNA Polymerase to function

41
Q

Describe the distal promoter.

A
  • found -500 to -1000bp away from TATAbox (-1)

- contains response elements.

42
Q

What are response elements? Give an example.

A

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.

43
Q

Define enhancer regions.

A
  • 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
44
Q

What is a clamp protein?

A

a protein that binds chromosomes to proteins of the nuclear matrix.

45
Q

Briefly describe how the different promoter types modulate transcription.

A
  • 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.
46
Q

How do co-activators enhance RNA-POL activity?

A
  • assist in the assembly of the pre-initiation complex

- alter the chromatin state by changing the histones so DNA is more accessible for transcription

47
Q

what is CBP and what is its function?

A

-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

48
Q

Where specifically does CBP operate?

A

CREB-binding Protein targets histones located in the proximal and core promoters.

49
Q

Explain the process of a functional Glucocorticoid Response Element.

A
  • 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.
50
Q

briefly explain what SW1/SNF is and what its function is.

A

Switch/Sucrose Nonfermenting, these molecule is an ATP dependent remodeling complex.
These molecules remove the histone in the core promoter region.

51
Q

what does TFIID do?

A

TFIID also uses histone acetyltransferase activity at the core promotor region.

52
Q

How would you figure out the function of a promoter? Explain the process

A

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
53
Q

What would happen if you deleted the TATA box from the core promoter?

A

you would get very little gene expression. only 38% of gene would be expressed.

54
Q

what would happen if you deleted base pairs -40 to -70 bp away from the +1?

A

gene expression would still be relatively high, about 95%.

55
Q

What would happen if you deleted the GC box in the proximal promoter?

A

you could get very little gene expression. only about 14%

56
Q

what would happen if you deleted the CAAT box in the proximal promoter?

A

only 50% of the gene would be expressed.

57
Q

what if you deleted a repression element?

A

the gene would be transcribed more. about 114%

58
Q

Why is GFP such a handy tool?

A

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.

59
Q

What does potency mean in reference to a cell?

A

A cell’s ability to differentiate into other cells.

60
Q

Define totipotency.

A

A cell’s ability to differentiate into any of our adult cell types.

61
Q

Name the different stages and potency a cell will undergo.

A

-embryonic stem cells -> differentiate into progenitor cell types (they are pluripotent) –> three germ layers endoderm, mesoderm, ectoderm –>adult stem cells (multipotent)

62
Q

Give examples of adult stem cells.

A

intestinal cells (only last a few days), blood cells (last days to months depending on type) , neurons (it takes years to grow another)

63
Q

What are adult stem cells capable of?

A
  • 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
64
Q

What is a master switch?

A

A transcription factor that controls many many other transcription factors.

65
Q

How do researchers manipulate master switches? Give an example.

A

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.

66
Q

Explain the general process of what happens when a stem cell divides.

A
  • 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
67
Q

Define cell memory.

A

A master switch will remain on in differentiated cells to maintain that state.

68
Q

How does a differentiated cell remain in its final state?

A

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)

69
Q

Do stem cells require cell memory as well?

A

Yes. Master switches maintain stem cells, and can also influence other stem cell states.

70
Q

What state is the DNA in when located inside a totipotent cell?

A

DNA mostly in a euchromatin state.

Except for Constitutive Heterochromatin

71
Q

How is the state of DNA effected by the differentiation of a cell?

A

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.

72
Q

Describe the theory/application of stem cell therapy.

A
  • 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.
73
Q

Where do researchers harvest stem cells from?

A
  • Totipotent embryonic stem cells (create an embryo just to remove the stem cells from it. slightly unethical)
  • Induce Pluripotent stem cells
74
Q

What is iPS? How does it work?

A

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.

75
Q

Generally speaking, what is transcriptional repression? Give an example.

A

Genes can be “turned off” by repressor TFs binding to the response elements associated with genes.
Example: Histone Deacetylases and Methyltransferases

76
Q

What is HDAC?

A

Histone DeAcetlyases Complex.

These remove acetyl groups from histones, causing DNA to wind more tightly around the histone. Silences gene activity

77
Q

What is Histone Methyltransferases? How does it work?

A

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.

78
Q

Is methylation reversible?

A

It is. however, methylated DNA often remains in that state during cell division.

79
Q

What is Maintenance Methyltransferase?

A

An enzyme that maintains the methylated state of the 5-methylcytosine bases in newly transcribed mRNA.

80
Q

How is methylated DNA maintained during cell division?

A

Maintenance Methyltransferase will travel along the newly transcribed mRNA and methylate the same cytosines that were methylated in the template strand.

81
Q

What types of DNA modifications remain in newly synthesized DNA?

A

Methylation and Histone Tail Modifications

82
Q

How do we know DNA methylation can be passed on to the next generation?

A

By using Illumina sequencing we can find where DNA methylation has occurred throughout the genome.

Bio 2520 (4 decks)

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