Gene Expression (I and II)

Question 1

What are the different components of a “gene unit”?

Answer 1

Insulator elements (i.e. barriers) on each end

Regulatory Elements

Proximal promoter region (-200 to +50)

Core promoter region (-40 to +40)

Coding region

Question 2

True or false: Regulatory elements are orientation and distance dependent

Answer 2

FALSE

Regulatory elements are orientation independent and distance independent.

If the elements are inverted, no difference in effect is observed

If the elements are moved closer or farther away, no difference in effect is observed

Question 3

How do regulatory elements interact with proteins?

Answer 3

Noncovalently

Question 4

How can regulatory elements thousands of base pairs apart all work together to regulate transcription?

Answer 4

Loops bring the regulatory elements all near to each other.

A mediator complex (>25 proteins) transmits effects of regulatory elements to the RNA polymerase

Question 5

What are the main features of regulatory elements?

Answer 5

Short (6-20 bp long) sequences of DNA that bind to transcription factors

Can be found up to 50 kB from start of transcription

Can be upstream or downstream

Each element can bind multiple transcription factors

Question 6

What are the different ways that chromatin can be opened up to induce transcription?

Answer 6

A transcription factor binds to a regulatory site on the chromatin

The following mechanisms all work together to cause transcription

Chromatin remodeling complexes (like SWI/SNF) can unwind chromatin

Histone chaperones can remove nucleosomes and replace them with specialized histone protein (ex: CENP-A)

Histone-modifying enzymes can produce a specific pattern of histone modification

Question 7

What do HAT and HDAC do?

Answer 7

HAT acetylates lysine residues, thus removing the positive charge present on the lysine side chain

HDAC deacetylates lysine residues

Question 8

True or false: The methylation state of lysine residues alters the charge of the lysine side chain and therefore whch proteins can bind at that residue.

Answer 8

FALSE

Methylation of lysine does not alter the charge on the risidue, but it does change which proteins can bind and interact at that residue

Question 9

How does acetylation lead to increased gene expression?

Answer 9

Acetylation opens up heterochromatin structure converting it to euchromatin which can be actively transcribed

1) It eliminates the ionic interactions between lysne (+) and DNA (-)
2) The acetyl group also provides a protein binding site

Question 10

What modification can occur at arginine residues?

Answer 10

Methylation

Dimethyl arginines can be either asymmetrical or symmetrical

Question 11

What is a chromodomain?

Answer 11

A protein domain that binds to methylated lysines and methylated arginines

Question 12

What is a bromodomain?

Answer 12

A protein domain that binds to acetylated lysine groups

Question 13

Describe the sequential process of writing and reading the histone code to regulate gene transcription.

Answer 13

1) Gene activator protein and HAT bind to regulatory element on DNA
2) HAT acetylates H3K9
3) Histone kinase phosphorylates H3S10
4) HAT acetylates H3K14
5) Chromatin remodelin complex binds 1 H3 and TFIID binds other H3 (Via bromomdomains)
6) Transcription machinery assembles leading to transcription

Question 14

What are the common motifs of DNA binding proteins?

Answer 14

1) Helix-turn-helix
2) Helix-loop-helix
3) Zinc finger
4) bZIP

Question 15

What are the different modules often found in transcription factors?

Answer 15

Activation domain

Dimerization domain

DNA binding domain

Question 16

Which groove of DNA do transcription factors primarily bind to?

Answer 16

Transcription factors primarily bind to the major groove of the DNA double helix

Question 17

Describe the helix-turn-helix motif

Answer 17

Composed of a recognition helix, which binds to the major groove of DNA, which has an edge specific to a particular nucleotide sequence, and a support helix

These motifs often dimerize with recognition helices 3.4 nm apart, allowing for binding at each turn of the helix

Dimers of this type always bind to palindromic DNA sequences

Question 18

What is a pseudopalindrome?

Answer 18

A DNA sequence that is palindromic at the ends, but has a series of nucleotides in the middle (corresponding with the minor groove of the DNA helix) that are not palindromic.

Question 19

Describe the helix-loop-helix motif

Answer 19

Can be homodimers or heterodimers

Composed of a recognition helix and a dimerization helix

The dimerization domain often is composed of leucine zuppers

Recognition helices from each monomer bind to adjacent turns of the major groove

Question 20

Describe the zinc finger motif

Answer 20

A zinc atom holds the structure together

Four “ligand bonds” between the zinc atom and 2H/2C or 4C produce the characteristic shape

Distance between ligand bond residues in this structure is usually: 2or3bp—-12/13bp—-2or3bp

Often joins alpha helix (recognition helix) and beta sheet together

Question 21

What gives zinc finger domains high affinity for specific DNA sequences?

Answer 21

Proteins typically have multiple zinc finger modules that recognize successive portions of the DNA sequence

Question 22

What is a bZip motif?

Answer 22

Scissor shaped dimer joined by leucine zipper composed of basic amino acids

Bind to palindromic DNA sequences

Question 23

What are some methods used to analyze regulatory elements in gene expression?

Answer 23

Footprinting with DNAaseI for transcription factor binding sites

Gel-mobility shift assay to show protein-DNA complexes

Chromatin immunoprecipitation (CHIP assay)

Reporter genes to assay regulatory elements in specific cells

Question 24

Describe the process of footprinting with DNAseI

Answer 24

This method allows us to determine where regulatory element may exist within a particular gene unit

DNA binding proteins will protect the DNA beneath from cleavage by nuclease.

Following random cleavage and separation of nucleotides by electrophoresis, a footprint where no cleavage is observed will be visible

Question 25

Describe a gel retardation experiment and what it shows us.

Answer 25

Used to examine protein-DNA or protein-RNA interactions

The basic idea is that DNA with proteins complexed onto it will move slower than those without proteins bound in a gel electrophoresis experiment.

Size and charge of the proteins will impact how the complex moves through the gel

Question 26

Describe the process of chromatin immunoprecipitation (CHIP)

Answer 26

DNA in living cells has regulatory proteins bound to the gene regulatory elements

These proteins are cross-linked to the DNA with formaldehyde

Cells are lysed and the DNA is broken into small fragments

Antibodies for a specific regulatory protein (that is now cross-linked to its DNA fragment) are used to precipitate out the DNA of interest

The only DNA left following precipitation will be the DNA that the regulatory protein was bound to

Crosslinks can be removed (with acid) and the DNA can be amplified for further analysis

Question 27

How do reporter genes work?

Answer 27

Reporter genes are used to study the role that enhancers play in gene regulation.

An enhancer of interest is inserted into a bacterial plasmid

This enhancer is used to enhance the activity of a reporter gene that is easy to assay (example: luciferase, beta-galactosidase)

If the reporter gene shows high expression, then the transcription factor was active in promoting the transcription of the gene of interest

Question 28

What is combinatorial gene control?

Answer 28

Multiple different regulatory proteins can bind to an identical regulatory element in a gene.

The combination of proteins that bind to an element depends on the cell type or physiological state of the cell

Some combinations will activate and some will repress gene expression

Question 29

Describe the different dimers that can form between Max, Myc and Mad

Answer 29

Max exists as a homodimer Max-Max

Max can also heterodimerize to form Myc-Max and Mad-Max

Question 30

How do the different dimers of Max, Myc and Mad affect gene transcription for cell division and differentiation?

Answer 30

These dimers bind to E-box elements of genes required for cell division and determine whether a cell will proliferate or differentiate

Max-Max binds the E-box and silences transcription (this is the normal state)

Myc is produced when cells want to divide, so Myc-Max dimers bind and upregulate the genes for cell division causing transcription.

Mad is produced when a cell wants to differentiate, so the Mad-Max dimers bind and repress the transcription of differentiation genes.

Question 31

What DNA binding motif are the Mad, Max and Myc dimers?

Answer 31

Helix-loop-helix

Question 32

In which stage of the cell cycle is Myc expressed?

Answer 32

Myc is expresed in the G1 to S transition of the cell cycle.

When cells want to divide.

Question 33

How does a kinase cascade lead to activation of transcription factors?

Answer 33

An extracellular signal (mitogen) activates a kinase.

This kinase then activates (terminal phosphate from ATP transferred onto the serine of a protein) another, which activates another … and so on.

The final kinase in the cascade is usually a nuclear kinase (MAPK) which is found on the nuclear membrane. When phosphorylated, the MAPK enters the nucleus and activates the transcription factor by phosphorylation

Question 34

How are Fos and Jun activated?

Answer 34

Fos is phosphorylated by ERK (a MAPK protein) and Jun is phosphorylated by JNK (a MAPK protein)

Question 35

Why is 1 X chromosome inactivated in females?

Answer 35

Dosage compensation: if no silencing occured, then expression of the genes found on the X-chromosome would be twice as high as that seen in males, which would be toxic

Question 36

Describe how females are mosaic due to X-chromosome inactivation

Answer 36

In the early embryo, each cell contains an active paternal and maternal X-chromosome

One of the X-chromosomes is randomly condensed and inactivated

All progeny cells from here will have the same x-chromosome inactivated as the parent cells

This leads to a mosaic in which patches of cells express each x-chromosome (Calico cat example)

Question 37

Describe how the globin gene cluser on chromosome 11 is regulated.

Answer 37

Different regulatory control proteins bind and induce opening of the globin gene at different positions depending on the stage of life.

This causes the different hemoglobin subunit types between conception, birth, and early childhood

Question 38

True or false: If two alleles have the same nucleotide sequence, they will have the same inheritable genetic information.

Answer 38

FALSE: epigenetics

Modification of DNA and regulatory proteins can lead to different inheritable genetic information from identical nucleotide sequences

Question 39

What are the 4 main types of epigenetic mechanisms?

Answer 39

1) Positive feedback transcription factor loops (self-activation)
2) DNA methylation
3) Acetylation and deacetylation of histones (interconvert between euchromatin and heterochromatin)
4) Histone phosphorylation and histone methylation

Question 40

Describe a positive feedback transcription loop.

Answer 40

Protein A is not made because it is required for its own transcription

A transient signal turns on expression of protein A, which then promotes the synthesis of more protein A

When the cell divides, protein A will be present, so A will be produced in the progeny as well.

Question 41

How does asymmetry within the embryo lead to cell differentiation?

Answer 41

In successive cell divisions, different regulatory proteins will exist in each daughter cell.

The presence of different combinations of transcription factors leads to cell differentiation.

After 2 divisions with 5 transcription factors, 8 cell types can form

With 25 regulator proteins, 10,000 cell types can form

Question 42

What sequences are methylated in DNA?

Answer 42

CpG sequences

These are palindromic

Question 43

Does methylation usually promote or inhibit gene expression?

Answer 43

Methylation usually inhibits expression of the gene

Question 44

True or false: Methylation of cytosine does not interfere with the formation of H-bonds.

Answer 44

True

The NH2, H and carbonyl O of the cytosine are all still available to participate in hydrogen bonding

Question 45

What enzyme ensures that methylation is transferred to progeny cells?

Answer 45

Maintenance methylases recognize hemi-methylated CpG’s following DNA replication and produces dimethylated CpG’s to match the parent DNA

Question 46

How can a gene be completely turned off due to methylation?

Answer 46

If gene regulatory proteins are lost from the DNA, the gene is off but leaky (expressed at a basal rate)

The exposed DNA can be methylated, which prevents RNA polymerase from binding properly

Proteins recognize the methylated DNA and recruit enzyme remodeling complexes and histone deacetylases which condense the gene and turn it completely off

Question 47

How do CG islands protect genes from methylation?

Answer 47

CG islands are found at the 5’ end of many housekeeping genes

They have repetitive CG sequences close together that actually protect the DNA from methylation rather than induce methylation like CpG bases found elsewhere

Question 48

What is the role of maintenance acetylases?

Answer 48

They act to acetylate histone proteins of nucleosomes that are hemi-acetylated in newly formed progeny cells. Preserves the acetylation patern of the parental cells

Question 49

What is genetic imprinting?

Answer 49

Certain genes are expressed in a parent-of-origin manner rather than following Mendelian genetic rules

A particular gene retains its methylation in either the sperm or the egg, but not both

Most genes lose their methylation during meiosis, but a small percentage do not