RR3: Gene Regulatory sequences

Question 1

What is the TATA box?

Answer 1

It’s a highly conserved sequence of TATA(a or t)A(a or t)(a or g) located at 30 nucleotides upstream (negative) of the start site. It directs transcription at the promoters of some protein-coding genes. It’s a cis-acting element driving transcriptional initiation.

Question 2

What are some other proximal cis-acting elements driving transcriptional initiation?

Answer 2

BRE (TFIIB recognition element)
TATA box
Inr (initiator)
DPE (downstream promoter element)

they don’t all come together necessarily and they are not present in all genes.

Question 3

What does a cis-acting element mean?

Answer 3

So CIS acting means that it’s within the DNA or the chromosome that’s actually containing the gene.

Question 4

TRUE OR FALSE. Most eukaryotic genes are regulated by multiple transcriptional control elements.

Answer 4

TRUE!!

Question 5

What are CpG island?

Answer 5

They’re the most common promoters in mammalian cells. They are rich in CGCGCGCGCG. They are used for transcriptional silencing of the gene.

They can also send RNA polymerase in both directions leading to divergent transcription.
They might be the cause of why 80% of the genome is transcribed.

Question 6

Is the genome of yeast more or less compact than the human genome, why?

Answer 6

The yeast genome is more compact because it has very few introns that are very small.

Question 7

Do all genes have tata box? why?

Answer 7

No. Usually, only genes that are actively transcribed with respect to a given change have a TATA box.

Question 8

Where are generally situated the proximal elements?

Answer 8

-200 to +1, but there can be some situated downstream in some cases.

Question 9

Do genes need a CpG island promoter and a TATA box to survive?

Answer 9

No. They can have one or the other, or both or none. They do need some type of promoter to initiate transcription, but not necessarily TATA box or CpG island promoter.

Question 10

Do yeasts have TATA box?

Answer 10

yes, usually. it’s at -90.

Question 11

What are enhancers?

Answer 11

They can act up to 50 kB away from the gene they are affecting. They can increase the transcription of genes.

They are necessary for interacting with key proteins that activate the transcription in specific tissues.

Question 12

How can enhancer influence their target gene when they are so far away?

Answer 12

Because genes would be in loops. It results in regions that are linearly very distant becoming physically adjacent to one another. So the enhancer and its target gene could be 50kb away from each other, but in a loop chromosome, they are next to each other.
Formation of topologically associated domains.

Question 13

What would be the size of a chromosome loop?

Answer 13

30nm chromatin fiber.

Question 14

What are these chromatin loops often associated with?

Answer 14

Active transcription.
Enhancers help generate, stabilize and increase the rate of transcription.
The loops change and evolve. Proteins are maintaining the loops under specific situations.

Question 15

TRUE OR FALSE. UAS is only for yeast. We don’t find it in humans.

Answer 15

TRUE!!!

Question 16

Why are bacterias used a lot in recombinant DNA technology?

Answer 16

Because bacteria can reproduce rapidly. So we use it to make a lot of DNA.
We can introduce DNA segments into bacteria and grow a lot of them. Then, we purify the DNA and we can use the DNA for downstream applications.

Question 17

Why are mammalian cells used in recombinant DNA technology?

Answer 17

Mammalian cells are used as a factory for the expression of DNA segments. So we can actually see what the genes do to the cell.

Question 18

What is recombinant DNA technology?

Answer 18

A method used to combine DNA to create genes with new functions.
We can create vectors that will be transfected in mammalian cells.

Question 19

What’s the gene TTR?

Answer 19

Transthyretin. Important transport protein that carries vitamin A and thyroxine.

Question 20

What are the steps to identify transcriptional regulatory regions?

Answer 20

1. Find the gene of interest Gene X
2. Take a piece of DNA that includes a common region like the TATA box or an initiator, but always the same in every DNA segment of gene X.
3. 5’ deletion. Using restriction enzymes or PCR reactions, cut the segments from the 5’ end to get small fragments.
4. We get a series of variants of the same gene, keeping the same 3’ end, but changing the amount of regulatory sequence on the 5’ end
5. We use a vector designed to express genes in mammalian cells and introduce those sequences in the vector
6. We end up with different vectors that we can transfect in mammalian cells
7. The reporter gene in the vector will give a readout on how effective the transcription was.
8. We can now tell in which sections where the important regions for transcriptions are.
9. Make conclusions about where the regulatory regions are.

Question 21

What are examples of reporter genes and what do they do?

Answer 21

GFP: fluorescent
Beta Galactoside (lacZ): blue color
Luciferase (luc): Gives off light
thymidine kinase (tk)
chloramphenicol acetyltrasnferase (CAT)
Help assess how effective the transcription was.

Question 22

What are linker scanning mutations?

Answer 22

Used to make variations of a gene. Using restriction enzymes on an important regulatory region of a gene, we cleave small sections and ligate it back together without that small section. In each variant, the sections are slightly overlapping, so we can match more precisely which sections are responsible for transcription. We can put those segments in a vector that has a reporter gene, so we can observe how effective the transcription was.
We can find proximal-promoter elements.

Question 23

Why is PAX6 an important gene?

Answer 23

Important for the development of eyes and PAX6 has enhancers specific to the retina

Important for the formation of a functional pancreas and PAX6 has enhancers specific to the pancreas.

Important for regions of the brain and PAX6 has enhancers specific for the brain.

So there are 3 different promoter that drive 3 different types of PAC6 mRNA.

Question 24

Are introns coding regions?

Answer 24

NO! Introns are not coding regions, but they are still important. There are enhancers that are in regions that give rise to introns. There’s still a lot of information in regions that encode introns.

Question 25

Is nucleotide sequence usually conserved? (in between organisms)

Answer 25

No. But some sequences are conserved.

Question 26

Why are some nucleotide sequences conserved?

Answer 26

Because they are absolutely necessary. There is something stopping them from changing. Most of the time, they are enhancers.

Question 27

Would it be possible to put a sequence of a human gene in a mouse?

Answer 27

Yes. If those sequences are highly conserved between those organisms, it is possible to transfect a human gene in a mouse. For example, an enhancer that was highly conserved could be introduced in a mouse and give rise to a viable mouse.

Question 28

Are genes and chromatin linear?

Answer 28

No. They form loops.

Question 29

In CpG island promoters, RNA polymerase 2 goes in both directions, which gives rise to transcription in both directions. Is the transcription as effective in both directions?

Answer 29

No. In the anti-sense direction, so 3’ to 5’ (the new strand is synthesized that way, with the 5’ last), the levels of transcripts are lower.

Question 30

Why is the normal sense of transcription more effective than the anti-sense?

Answer 30

Because there’s more polyadenylation signals in the anti-sense. So it gives rise to very short RNA transcripts.
There’s also very few U1 snRNP sites in the anti-sense transcripts. U1 snRNPs protect the mRNA from premature termination.
These 2 factors cause the anti-sense to have short mRNA transcipts that are degraded by the cell, thinking they’re useless.