FTM 42-43 - Gene Regulation

Question 1

At what level does the majority of gene regulation take place?

Answer 1

The transcriptional level

Question 2

What is an operator?

Answer 2

A region of DNA, in prokaryotes only, upstream and adjacent to the promoter that serves as a binding site for proteins that help regulate the gene expression.

Question 3

Define activator, silencer, enhancer, and repressor.

Answer 3

Enhancer: a short region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur.

Silencer: a DNA sequence capable of binding transcription regulation factors, called repressors.

Activator: a protein that binds to enhancers or other promoter-proximal elements to aid in the initiation of transcription

Repressor: a protein that binds to silencers or other promoter-proximal elements to inhibit transcription

Question 4

What are the proteins produced by the lac operon? What are their functions?

Answer 4

Question 5

Draw a map of the lac operon and describe what each part is for?

Answer 5

Question 6

List and describe the function of all of the lac operon elements.

Answer 6

• lacZ* - gene for ß-galactosidase
• lacY* - gene for permease
• lacA* - gene for transacetylase
• lacP* - promoter for lacZ, Y, & A
• lacO* - the operator for lacZ, Y, & A
• lacI* - gene for repressor protein

Question 7

What is an inducible gene?

Answer 7

A gene whose expression is either responsive to environmental change or dependent on the position in the cell cycle

Question 8

What is the lac operon repressor protein and what are its functions and when?

Answer 8

The protein is called the “lac repressor” and it functions by binding to the the lacO region of the of the operon so that RNApol cannot behind to initiate transcription. It does this when lactose is absent.

When lactose is present, an isomer of lactose, allolactose, is also present. Allolactose will bind to the lac repressor causing it to fall off of the lac operon.

Question 9

What are the mutations affecting the lac operon and how do they affect it?

Answer 9

Question 10

What does it mean when it is said that a genetic element is under positive or negative control?

Answer 10

Question 11

How does the presence of glucose affect the lac operon? How does it do this?

Answer 11

If lactose is not present then the presence of glucose has no significant effect; the operon is still off. However, if lactose is present then the presence of glucose will prevent the operon from being maximally efficient. This is because the lac operon genes need an activator bound to the promoter to reach a maximal txn level. When glucose is present this activator cannot bind the lac promoter.

Question 12

How does the absence of glucose affect the lac operon? How does it do this?

Answer 12

If lactose is not present then the absence of glucose has no significant effect; the operon is still off. However, if lactose is present then the absence of glucose will help to activate the lac operon. This is because the lac operon genes need an activator bound to the promoter to reach a maximal txn level. When glucose is absent, cAMP is high and cAMP will bind CRP or CAP (cAMP receptor protein or catabolite activator protein). This complex can then bind lacP to help activate txn.

Question 13

Answer 13

Question 14

Answer 14

Question 15

Broadly define “cis regulatory element” and “trans regulatory element.”

Answer 15

Question 16

What are the three cis regulatory elements we need to know?

Answer 16

Question 17

Which two cis regulatory elements are found within the basal promoter region?

Answer 17

TATA and CAAT boxes

Question 18

What are the three protein domains commonly seen in trans regulatory elements?

Answer 18

Question 19

List and describe the three modes of action of repressors?

Answer 19

Question 20

T/F - a cis regulatory element can regulate many different genes?

Answer 20

True, this provides a mechanism to coordinate gene regulation of many genes at once

Question 21

What is a response element?

Answer 21

A short sequence of DNA within a gene promoter region that is able to bind a specific transcription factor and regulate txn of genes.

Question 22

Describe the hypoxia response mechanism.

Answer 22

Under normal conditions, the txn factors HIF-1α and HIF-1ß (ARNT) are constitutively expressed with HIF-1ß residing in the nucleus and HIF-1α being hydroxylated by prolyl hrdroxylase (using O2) which targets it for ubiquitination and degradation. In the absence of O2, prolyl hydroxylase cannot hydroxylate HIF-1α and it translocates into the nucleus where it dimerizes with ARNT and binds to the hypoxia response elements (HREs) at 5’-TACGTG-3’ of several genes that will enhance O2 delivery to tissues and/or energy supply via glycolysis.

NOTE - THIS IS NOT AN OPERON, IT IS A FAMILY OF GENES HAVING THEIR EXPRESSION INDUCED BY TXN FACTORS BINDING TO SIMILAR ENHANCER SEQUENCES

Question 23

Differentiate enhancer from response element.

Answer 23

A response element is within a gene promoter. A enhancer is not

Question 24

Why is the hypoxia response element being researched as a target for cancer treatment?

Answer 24

Question 25

Why type of compound is a glucocorticoid receptor (GR)? What does it do?

Answer 25

It is a zinc-finger type txn factor. When it is bound to a glucocorticoid, forming a GR complex, it binds to hormone response elements and up regulates the expression of anti-inflammatory genes in the nucleus and represses the expression of of pro-inglammatory proteins in the cytosol.

Question 26

Describe the basic sequence of a hormone response element.

Answer 26

It is an inverted repeat separated by by any nucleotide sequence.

Question 27

Describe how GR is regulated and how it translocates into the nucleus.

Answer 27

GR without a glucocorticoid is held in the cytoplasm by several hsp proteins as part of an inactive multi-protein complex. When a glucocorticoid binds to GR it dissociates and then dimerizes with another GR complex. This GR complex dimer then translocates into the nucleus.

Question 28

Describe the Myc/Max system.

Answer 28

Myc has a txnal transactivation domain but it cannot bind DNA without dimerizing with Max. Max can dimerize with itself and bind enhancer DNA but this inhibits txn. Therefore, gene activation only occurs when both Myc and Max are expressed at the same time. This system regulates genes involved in cell cycle progression and proliferation. Both genes are only expressed when the cell is ready to proliferate.

Myc overexpression is often seen in tumor cells

Question 29

What are the two general types of regulation by RNAi?

Answer 29

1. Inhibition of tln on the ribosome - miRNA
2. Degradation of the target mRNA - siRNA

Question 30

Describe how miRNAs operate from formation to function.

Answer 30

1. An miRNA gene is transcribed producing long pri-miRNAs which are processed to pre-miRNAs hairpins by Drosha
2. Pre-miRNA is transported to the cytoplasm by Exportin 5
3. Dicer processes the pre-miRNA to ssRNA and initiates the formation of the RNA-induced silencing complex (RISC)
4. The miRNA in the RISC will base pair imperfectly with a sequence element in the 3’-UTR of a specific mRNA, preventing interaction of the tlnal machinery with the 5’ cap structure.

Question 31

Describe how siRNAs operate from formation to function.

Answer 31

1. Dicer converts a dsRNA into a siRNA duplex and initiates the formation of the RNA-induced silencing complex (RISC)
2. siRNA in the RISC perfectly binds to a sequence element in the 3’-UTR of a specific mRNA
3. Perfect pairing of siRNA with mRNA within the RISC will activate RISC endonuclease activity and the mRNA will be degraded

Question 32

Things to know about RNAi and cancer

Answer 32

Question 33

Things to know about forced RNAi

Answer 33

• Two opposable promoters in a gene delivery vector can be introduced into a cell so the dsRNA is transcribed
• The cells own dicer will then process that dsRNA into siRNA and degrade a target mRNA
• This method can be used to silence specific genes
• The could be applied to HIV, Flu, Cancer, and many genetic diseases