Lecture 19 - Cis-Acting Regulatory Elements in RNA Polymerase II Promoters

Question 1

In mammalian cells, where does the TATA box usually sit?

Answer 1

Somewhere Between -26 and -31

Question 2

What does the initiator element (Inr) do?

Answer 2

The initiator element plays some role in appropriately positioning and enhancing the efficiency of initation, but it’s not as important as TATA.

Question 3

Where is the initator element (Inr) usually found?

Answer 3

It is usually situated around the transcription start site, where usually there is a C at the -1 site and an A at the +1 site.

Question 4

What is the TFIIB recognition element (BRE)?

Answer 4

The BRE is the site where TFIIB interacts with the DNA.

Question 5

What do the downstream promoter elements (DPE) do?

Answer 5

The DPEs contribute to transcriptional efficiency.

Question 6

How much of the genome is transcribed?

Answer 6

80%

Question 7

With promoters that work in both directions, what kind of transcripts can be formed?

Answer 7

Sense transcripts are formed, but very often, antisense transcripts can also be formed.

Question 8

Where are divergent transcripts usually prevalent?

Answer 8

Divergent transcripts are usually prevalent on promoters that have CpG (C phosphate G) islands and those that do not have TATA boxes.

Question 9

What is divergent transcription?

Answer 9

Divergent transcription is when both sense and antisense transcripts are produced by reading on both sides of the promoter.

Question 10

Why do CpG regions give rise to divergent transcription?

Answer 10

CpG regions can be read from either side of the double stranded DNA, so polymerase will join on both sides.

Question 11

Which type of transcript is made more in divergent transcription? Why might this be?

Answer 11

Sense

This may be because of the presence of splicing sites in the antisense direction, which may give rise to an elongation problem, or disrupt transcript stability.

Question 12

What are promoter-proximal elements? What do they do?

Answer 12

Promoter-proximal elements are other conserved sequences that are important for the activation of specific genes. They may be recognized by specific proteins that interact with DNA and enhance transcriptional efficiency of the downstream genes.

Question 13

Where are enhancers found?

Answer 13

Enhancers tend to work distally in promoters, far from the transcription start sitres. They can be kilobases upstream or downstream of the gene that they are affecting.

Question 14

What is the difference in directionality between promoter-proximal elements and enhancers?

Answer 14

Enhancers work in a non-directional way. You can flip them around and they are equally effective. This is not the case for promoter-proximal regions.

Question 15

What do yeasts have instead of enhancers? Why?

Answer 15

Yeast genes have very few introns and the TATA boxi s present around -90 (quite far from the transcription start site). Instead of having enhacers, they have upstream activating sequences (UAS), which are upstream of the TATA box in these gemes but often not as far away as the enhancers in mammalian genes.

Question 16

What does mammalian genes having distant regulatory elements suggest?

Answer 16

Mammalian genes having distant regulatory elements suggests that they are involved in setting up topological domains in the chromatin, which brings them in proximity to the given promoters of the gene that they are affecting.

Question 17

What does the PAX6 gene in humans play a role in?

Answer 17

Development of the Eyes

Question 18

What do enhancers very often respond to?

Answer 18

Enhancers very often respond to DNA-binding proteins that are specific to given tissues. These are called tissue/temporal-specific DNA-binding transcription factors.

Question 19

How can you use recombinant DNA technology to analyze what the proximal region does to transcriptional output?

Answer 19

You can use recombinant DNA technology in order to make plasmids that have a series of deletions that you can generate in a 5’ to 3’ manner, where you whittle down the proximal region of a given control region of a gene, then analyze what the pertubations in that proximal region do to the transcriptional output of that target gene (via looking at a reporter gene). In order to get the plasmids into mammalian cells, they are amplified in bacteria and then transfected into mammals.

Question 20

What are transformation, transfection, and transgenics?

Answer 20

Transformation involves the insertion of DNA into a specific vector that can grow in bacteria. It allows DNA amplification and purification. Transfection and transgenics involve the introduction of similar vectors into mammalian cells and into animals or plants (respectively).

Question 21

How can you whittle down the proximal region of a given gene?

Answer 21

You can make increasingly smaller, overlapping PCR constructs of the proximal region.

Question 22

What are reporter genes?

Answer 22

Reporter genes are genes that allow you to assess gene expression.

Question 23

GFP, β-galactosidase, thymidine kinase, chloramphenicol, and luciferase are all examples of what?

Answer 23

Reporter Genes

Question 24

Which regions of the genome do and don’t have variation between organisms?

Answer 24

There’s a large amount of variation between organisms in the nucleotide sequences of the regions that don’t necessarily have important consequences downstream. In regions where any change will increase change of death, there’s very clear selection against changes in those regions.

Question 25

What does synteny refer to?

Answer 25

Often, synteny refers to the conservation of blocks of order within two sets of chromosomes that are being compared with eachother.

Question 26

One way of assessing proximal regions of genes is using recombinant DNA technology. What is another way they can be assessed?

Answer 26

Proximal regions can be assessed by comparing genome sequences between organisms since the promoter region nucleotide sequences are highly conserved.

Question 27

What can you assess by introducing specific types of recombinant DNA molecules containing regulatory elements into an organism?

Answer 27

This allows you to assess what those regulatory elements might do regarding transcription of a given reporter gene.

Question 28

What is the main difference between recombinant DNA techniques and linker scanning mutations?

Answer 28

Both techniques have parts of the upstream portion cut out, but recombinant DNA techniques has progressive cuts made in the same direction while linker scanning mutations have overlapping regions being removed from anywhere in the segment. Basically, if you have segments of 4k, 3k, 2k, and 1k bp made via recombinant DNA techniques, all the shorter ones than 4k will be missing the same 1k bp, and all the ones shorter than 3k will be missing the same additional 1k bp and etc.. Meanwhile, linker scanning has all segments of the same length, just with a different portion removed from anywhere in the segment.

Question 29

What does putting regulatory sequences upstream of a β-galactosidase gene allow you to do? What organisms are the regulatory elements defining where and when the gene is going to be expressed conserved in?

Answer 29

This allows you to colour tissues with X-gal.

Frog, Fish, Mouse, and (probably) Humans

Question 30

What process can we use to identify the proteins that interact with regulatory sequences?

Answer 30

Electrophoretic Mobility Shift Assays (EMSA)

Question 31

What are EMSAs good for?

Answer 31

Electrophoretic mobility shift assays are good for identifying DNA binding transcription factors that interact with a given sequence you are interested in.

Question 32

How are electrophoretic mobility shift assays used to identify DNA binding transcription factors for a sequence of interest?

Answer 32

A stable DNA-protein complex is formed that can be identified using a labelled DNA probe that will light up where you see the event taking place. The sample is run through a non-denaturing polyacrylamide gel to separate the bound probe from the free probe, showing you what portions of your sample have the DNA-protein complex.