a. Tutorial 1: Gene Regulation

Question 1

Describe gene regulation

Answer 1

the control of gene expression

Question 2

Describe gene expression

Answer 2

the process in which the info encoded in a gene is converted into an observable phenotype

Question 3

What is the significance of the TATA box? What is its position in the genome?

Answer 3

a) it is the site where the pol binds to the DNA
b) approx 30bp upstream of the start site (-30)

Question 4

Describe transcription factors. What is the importance of promoter elements wrt transcription factors?

Answer 4

a) proteins that bind to DNA in order to regulate transcription
b) they provide a binding site for these factors

Question 5

What is the fxn of the following transcription factors? How do they do this?
a) repressors
b) activators

Answer 5

a) slow down the rate of transcriptions by binding repressor sequences
b) speed up the rate of transcription by binding enhancer sequences

Question 6

T or F - repressor/activator GTFs can only regulate gene transcription upstream of the site of the start just like the core promoters.

Answer 6

F - can be be located either upstream or downstream

Question 7

Describe a regulatory gene. What does this type of gene produce?

Answer 7

a) a gene on the DNA that is responsible for regulating another gene more downstream on that same DNA
b) produces a regulatory protein that binds onto a regulatory sequence (in promoter) in order to regulate the transcription of a target gene

Question 8

Draw a flow chart of gene regulation b/w bacteria and eukaryotes.

Answer 8

Question 9

Fill in the blanks; The promoter sequence in a eukaryotic cell is __________ of the start site. This sequence contains the ___________ which acts as the binding site for the RNA pol II.

Answer 9

upstream, TATA box

Question 10

T or F - All regions within DNA code for different proteins

Answer 10

F - some are non-coding = junk DNA

Question 11

In order to activate transcription there are GTFs that are bound to both the enhancer sequence upstream of the promoter sequences. As well as GTFs that bind to the promoter sequnce. However, even after this occurs transcription does not start. What else needs to occur for transcription to be initiated?

Answer 11

the DNA needs to fold in on itself in a way that allows the GTF bound to the enhancer to directly interact w/ the GTFs on the promoter sequence. This will jumpstart the transcription of the gene.

Question 12

Draw a DNA diagram that includes the following. Indicate where the pol II + the transcriptional factors would bind
a) an Enhancer
b) a core promoter
c) a repressor
d) a target gene

Answer 12

The transcriptional factors will bind to the core promoter, enhancer, and repressor sequence. While the RNA pol II will bind to the Core promoter after the transcriptional factors are in place.

Question 13

What is the significance of a Reporter Plasmid?

Answer 13

a tool used to determine the specific regions of a promoter that are associated w/ gene regulation as well as finding their fxn

Question 14

What is used to construct a reporter plasmid? How does this tool work?

Answer 14

DNA cloning = is a tool used to study DNA regulation (and other features) by inserting a sample DNA into a plasmid and having it undergo transformation into a bacterial cell

Question 15

Describe a reporter gene. Does it contain the promoter sequence of interest?

Answer 15

the gene that is directly under the control of the promoter of interest (therefore no). It codes a protein w/ an easily measurable activity

Question 16

What are two types of proteins that would make great reporter proteins? What do they produce?

Answer 16

1. luciferase - produces light (bioluminescence)
2. green fluorescent proteins (GFP) - fluorescent microscope

Question 17

What is a reporter plasmid made of?

Answer 17

1. reporter gene - reports the expression of the protein
2. plasmid - ring in bacteria that contain the reporter gene
3. promoter of interest - engineered regulator sequence inserted into plasmid upstream of the reporter gene

Question 18

How does one test the activity of a promoter sequence in different conditions?

Answer 18

they insert it in a reporter plasmid and use the reporter gene to report the expression of the protein after the reporter plasmid is transfected into different cells. this exposes the plasmid to different conditions.

Question 19

What is the purpose of deletion mapping experiments? How does it do this

Answer 19

a) used to determine the sequence w/in the promoter that is the most critical for transcription
b) you start deleting certain sequences w/in the promoter and then compare its activity w/ a complete promoter. The deletion that has the biggest influence on the transcription activity the most is the most critical

Question 20

In the 6th plasmid depicted in the deletion mapping experiment they found that deleting the region b/w -130 and -110 resulted in an INC in transcription. What does this mean?

Answer 20

that that region is most likely a repressor sequence as they tend to reduce transcription.

Question 21

What are the 7 steps associated with Chromatin immunoprecipitation?

Answer 21

1. given a chemical that cross-links the transcription factors (TFs) to the open chromatin
2. shear the chromatin into fragments (TFs are still cross-linked)
3. incubate the fragments w/ an antibody that targets a specific TF.
4. the chromatin fragments that are cross-linked to the targeted TF contain a large bead that causes it to precipitate (isolates it from non-target TFs)
5. Reverse the cross-linkage of the target TF and isolate it from the chromatin
6. amplify the target chromatin fragment via PCR (using engineered primers)
7. Run PCR products through gell electrophoresis and compare the bands to the ladder.

Question 22

What is the purpose of ChIP (chromatin immunoprecipitation?

Answer 22

to use Transcription factors that bind onto certain promoter sequences to amplify and study certain promoter sequences of interest

Question 23

What will happen if the core promoter of a gene is mutated such that the GTFs cannot bind?

Answer 23

RNA pol II will be unable to bind. Resulting in no transcription or expression of the gene

Question 24

You use deletion mapping and a reporter plasmid assay to study the promoter of the gene. When you delete an element you notice an increase in the expression of the reporter protein. What type of element was deleted?

Answer 24

Repressor sequence

Question 25

Based on in vitro experiments you suspect a transcription factor binds a regulatory sequence of a gene, so you perform ChIP to confirm if this happens in vivo. 3. After running the PCR products on an agarose you expect a band around 100 bp based on the specific primers you designed for that sequence, but when the dye gel is stained there is no band in control lane or the sequence of interest lane. What does this tell you about the protein-DNA interaction?

Answer 25

The protein does not bind that regulatory sequence in vivo.