Lecture 4

Question 1

What is differential gene expression?

Answer 1

The process by which cells become different from one another by expressing unique combinations of genes.

Question 2

Three postulates of differential gene expression.

Answer 2

1. DNA of all somatic cell nuclei of an organism contain the complete genome that was established in the fertilized egg.
   (Genomic equivalence)
2. The unused genes in each cell are not destroyed and they retain the potential of being expressed.
3. Only a small percentage of the genome is expressed in each cell, and a portion of RNA synthesized in each cell is specific for that cell type.

Question 3

What are housekeeping genes?

Answer 3

Genes expressed in every cell (genes for ribosomes and genes for RNA pol II)

Question 4

Two organisms studied gives evidence for genomic equivalence. What are they and what evidence do they give?

Answer 4

-Drosophila: chromosomes observed under microscope that showed that not all genes were being transcribed at once, and the activation of certain gene was dependent on the timing of development.

-Dolly the sheep: one cell has capacity + info to differentiate into every other cell type in body (nucleus from differentiated cell into no DNA oocyte)

Question 5

Gene expression can be modified at 4 different step. What are they?

Answer 5

-Transcription (DNA -> RNA)

-pre-mRNA processing (pre-mRNA -> mRNA)

-translation (RNA -> protein)

-Post-translational modifications (modifications that regulate protein stability and/or function)

Question 6

What is a chromatin?

Answer 6

DNA strands are wrapped around histones which form chromatin.

Question 7

What are nucleosomes? Give characteristics.

Answer 7

Basic unit of chromatin.

Composed of his tone octamer.

Histone tails are positively charged.

The negative charge of DNA backbone neutralizes histones.

Question 8

What are the two types of chromatins? Which is loose and which is tight?

Answer 8

-Hererochromatin: tightly bound
-euchromatin: loosely bound

Question 9

Which type of chromatin proteins cannot bind to? How does that help.

Answer 9

Heterochromatin. So regions of DNA that shouldn’t be expressed are heterochromatin.

Question 10

In what way do transcription and pre-mRNA processing happen at the same time?

Answer 10

-pre-mRNA contains introns that haven’t been spruced out yet.

-while transcription happens, RNA pol II makes pre-mRNA

-at the same time, proteins at 5’ end of pre-mRNA are adding 5’ cap

-at the same time, enzymes add poly-a tail at 3’ end

-then: mRNA goes into cytoplasm

Question 11

What is 5’ cap?

Answer 11

Methylated guanosine place in opposite polarity of RNA.

This forms phosphate-phosphate bonds instead of phosphate-hydroxy bonds (so there is no free phosphates)

This prevents anything from degrading 5’ end.

Question 12

What is a poly-a-tail?

Answer 12

Sequence of adenosine that is added at the end of mRNA.

It stabilizes the pre-mRNA
The length dictates the stability

Question 13

What is a promoter?

Answer 13

Where RNA polymerase II binds to the DNA.

Some promoters have a TATA DNA sequence which is bound by the TATA-binding protein (TBP) which helps RNA pol II bind the promoter.

Question 14

What is transcription initiation site

Answer 14

where transcription begins, formation of the 5’ cap on RNA

Question 15

What are exons and introns?

Answer 15

Exons: regions of DNA that code for aa that will be part of the protein

Introns: non-coding regulatory regions that are spliced out of pre-mRNA and are not included in mRNA. Introns must be removed before the nature mRNA exits the nucleus.

Question 16

What is the translation inition codon?

Answer 16

ATG on the DNA

AUG on RNA

it’s the site where translation of the protein begins. Same ATG codon in every gene.

Question 17

What is 5’ UTR (leader sequence)

Answer 17

UTR = untranslated region

The sequence of base pairs between the transcription initiation start site and the translation initiation site. Often contains regulatory DNA elements.

Question 18

What is the translation termination codon?

Answer 18

TAA, TAG, or TGA on DNA

UAA, UAG or UGA on RNA

it signals the end of translation and the ribosome dissociates from the mRNA after encountering this codon.

Question 19

What is 3’ UTR?

Answer 19

The sequence of base pairs between the translation termination codon and the end of transcription.

Contains the polydenylation site (AATAAA), which is needed for polyA tail insertion.

Contains many cis-regulatory elements that determine mRNA stability and translation.

Question 20

What is the transcription termination sequence?

Answer 20

Termination continues beyond polyadenylation site for about 100 nucleotides before being terminated.

Question 21

What are CpGs?

Answer 21

C = 5’ cytosine

p = phosphate bond

G = 3’ guanosine

Question 22

What is a CpG island?

Answer 22

Regions of DNA where there is higher than expected frequency of CpG.

Question 23

What is observed-to-expected ration of CpG in humans?

Answer 23

GC content in DNA is 42% (21% + 21%)

Expected frequency is thus 4.41% (0.21 x 0.21)

Observed frequency is 1/5th of expected frequency.

Question 24

Why are CpG islands important?

Answer 24

They are important for transcription regulation because C can be methylated, which impacts RNA pol ll binding.

Question 25

Cis-regulatory vs trans-regulatory elements.

Answer 25

Cis regulatory = elements located on same chromosomes. Trans regulatory = different chromosomes

Question 26

Non-coding regulatory elements Enhancers vs silencers

Answer 26

Enhancers: promote gene expression Silencers: prevents gene expression Can be located far away from gene they control. Looping interaction with regulatory proteins bridges interaction.

Question 27

What are basal/general TFs?

Answer 27

group of proteins that bind every promoter in the genome, necessary for transcription to occur

Question 28

What are transcription factors (TFs)?

Answer 28

heterogenous group of proteins that bind specific DNA sequences in enhancers and promoters

Question 29

How do TFs explain gene expression specificity?

Answer 29

Within given cell, not all 1500 TFs will be expressed. Specific TFs will be expressed, which will bind to specific engenders ti activate specific genes. (Same thing for silencers)

Question 30

How do TFs promote gene expression?

Answer 30

Recruits protein to open chromatin. Allow looping (making enhancers more accessible) (TFs can also silence gene expression with the help of silencers)

Question 31

Explain specificity between TFs and enhancers. (3 points)

Answer 31

All cells have every enhancer, but not every TFs are expressed, thus not every enhancer is expressed. Some genes have multiple enhancers, which allowed it to be expressed in several different tissues but not others. One enhancer have multiple binding sites for multiple TFs (this allows more specificity. This prevents/protects against mutations (if TFs mutated or if enhancer is mutated)

Question 32

Enhancers have variable sequences, making them difficult to identify. So how do we do it?

Answer 32

Reporter assay -> two types GFP reporter assay: -Bind presumptive enhancer sequence to promoter + GFP gene -Is GFP activated? LacZ reporter assay -Codes for B-galactosidase -Check if B-galactosidase is transcribed (by checking for enzyme activity in certain tissue) -needs to be fixed

Question 33

Example of silencer seen in class

Answer 33

NRSE (neural restrictive silencer element) in mice -Prevent a promoter’s activation in any tissue except neurons. -Bound by a protein called neural restrictive silencer factor (NRSF) -NRSF is expressed in every cell that is not a mature neuron.

Question 34

Two mechanisms regulated transcription

Answer 34

-TFs -Epigenetic modifications

Question 35

What is epigenetic modification?

Answer 35

Epigenetics: refers to the study of gene expression changes that do not involve changes to the DNA sequence. These are modification to DNA that regulate whether genes are turned on or off.

Question 36

Two types of epigenetic modifications

Answer 36

-acetylation: opens up histones -methylation: histones or DNA

Question 37

Acetylation

Answer 37

Histone acetyltransferases place acetyl groups on histones. Acetyl groups are negatively charged. This negative charge repels negatively charged DNA, opening up the chromatin.

Question 38

What catalyses histones methylation?

Answer 38

-catalyzed by histone methyltransferases

Question 39

Does histone methylation enhance or prevent transcription?

Answer 39

-Depending on which lysine and which histone is methylated, histone methylation can enhance or prevent transcription. -Enhancement or repression also depends on the histone modifications in the vicinity, as multiple modifications are often found on the same gene.

Question 40

DNA methylation There are two types of promoters based if CpG content.

Answer 40

High CpG-content promoters (HCPs) and Low CpG-content promoters (LCPs)

Question 41

What are HCPs?

Answer 41

•Housekeeping genes (expressed in all cell types) •Developmental control genes (genes that regulate synthesis of TFs and regulatory proteins used in the construction of the organism) •Default state of these promoters is ON •To be turned off: histone methylation •These CpG islands are not usually methylated

Question 42

What are LCPs?

Answer 42

• Genes whose products characterize mature, differentiated cells (e.g. globins of red blood cells, hormones of pancreatic cells) •Default state of these promoters is OFF •The Cs in these promoters are methylated and this methylation is critical in preventing transcription •To be turned on: DNA methylation at the CpG sites is removed

Question 43

What does DNA Methylation do? How? (2 ways)

Answer 43

-blocks transcription 2 ways: -methylated C can recruit proteins that facilitates chromatin modification (MeCP2 recruits histones deacetylases + histones methyltransferase to create hetrochromatin) -methylation of C can physically prevent TFs from binding to DNA

Question 44

How does DNA maintain the methylated state?

Answer 44

MeCP2 also recruits a DNA methyltransferase called Dnmt3 which catalyzes de novo methylation -> spreading To maintain DNA methylation during cell division, an enzyme called Dnmt1 recognizes the methylated cytosine on the parental strand of DNA and methylates the daughter strand.

Question 45

What are imprinted genes?

Answer 45

Genes that are only active when inherited from mother (and not father) or father (and not mother). Imprinted genes are silenced by DNA methylation on CpG sites in promoters. In genes that are paternally imprinted, the DNA will become methylated in all sperm. In genes that are maternally imprinted, the DNA will become methylated in all oocytes.

Question 46

What is uniparental disomy? (UDP)

Answer 46

Happens when person received 2 genes from same parent (meiosis error) If gene is imprinted (from parent that it received from), then person miss this essential gene. This causes Beckwith-Wiedemann Syndrome.

Question 47

What is Beckwith-Wiedemann Syndrome?

Answer 47

Affected infants are larger than normal, and some may be taller. Children with the syndrome are at increased risk for some cancers. Most children and adults with the syndrome have a normal life expectancy. A group of genes on chromosome 11 are maternally imprinted. In children with this syndrome, they’ve inherited 2 copies of chromosome 11 from their father and none from their mother. This leads to them expressing too much of these specific genes, leading to overgrowth.