Control of Gene Expression; Eukaryotes 2

Question 1

What must binding to regulatory sequences be?

Answer 1

must be stronger than non-specific DNA binding

Question 2

What is NFkB?

Answer 2

• A rapid-acting primary transcription factor complex
• Responds quickly to stimuli e.g. antigens, UV light, free radicals
• Regulates many genes associated with innate and acquired immune system

Question 3

Do all cells have the same combinations of Transcription factors?

Answer 3

No, Different Cell Types have Different Combinations of TFs

Question 4

How are microarrays created?

Answer 4

1. isolate mRNA
2. Use reverse transcriptase to make cDNAs labelled with fluorescent dyes
3. Combine cDNAs and hybridise to DNA microarray

Question 5

What does each cell have?

Answer 5

Each cell type has a ‘signature’

Question 6

What is a ‘signature’?

Answer 6

Different patterns of gene expression from one cell to another, no one is the same

Not always the same as the proteins produced

Many additional levels of regulation

Question 7

What is post-transcriptional regulation?

Answer 7

After RNA polymerase has bound to the gene promoter and RNA synthesis has started

Modulates level of expression and can produce variant proteins

Question 8

What is alternative RNA splicing?

Answer 8

Method of increasing number of proteins expressed from a single gene

Up to 90% of human genes are expressed as alternatively spliced mRNA!

Can also have alternative 3’ splice sites, mutually exclusive exons or different combinations

Question 9

What is the Alternative Splicing of Fibronectin Gene? (1)

Answer 9

Adhesive protein that is secreted and binds other proteins together
Gene has multiple repeating exons. More than 20 isoforms made

Question 10

What is the Alternative Splicing of Fibronectin Gene? (2)

Answer 10

Exons EIIIA and EIIIB encode regions that bind to fibroblast plasma membrane
Adheres fibroblasts to extracellular matrix
Splice variant expressed in hepatocytes lacks EIIIA and EIIIB

Question 11

What proteins are involved in iron metabolism?

Answer 11

Transferrin – transport protein which carries iron in serum

Transferrin Receptor – membrane protein that binds iron-loaded transferrin and allows it to enter the cell

Ferritin – highly efficient iron-storage protein found in liver and kidneys

Question 12

How does high and low iron content, indicate to which protein we need more of?

Answer 12

High iron – need more ferritin and less transferrin receptor

Low iron – need less ferritin and more transferrin receptor

Question 13

Where is Ferritin expression controlled?

Answer 13

the Translational Level

Question 14

What does Ferritin mRNA have that contains iron?

Answer 14

has a stem-loop structure called an iron-response element (IRE)

Question 15

What does iron-response element do in response to low iron?

Answer 15

In low iron concentration this binds to an IRE-binding protein (IRP)
This blocks initiation of translation

Question 16

What happens in the expression of Ferritin when iron levels are high?

Answer 16

Binding of IRP to iron prevents binding to the RNA

Translation of ferritin which can bind excess iron

Question 17

How is Transferrin receptor controlled?

Answer 17

Need expression to be increased in low iron concentration

Transferrin receptor mRNA has iron response elements (IRE) in the 3’ untranslated region

Question 18

How is Transferrin expressed in low iron levels?

Answer 18

IRP protein binds to the IREs in low iron concentration
Translation can be initiated
Bound IRP helps protect the mRNA from degradation

Question 19

How is Transferrin expressed in high iron levels?

Answer 19

IRP cannot bind to the mRNA in high iron concentration
The transferrin receptor mRNA is rapidly degraded
The transferrin receptor is not expressed

Question 20

What are microRNAs?

Answer 20

Large class of regulatory RNA molecules
Are cleaved into small fragments of about 20 nucleotides long
Bind to mRNA molecules with complementary sequence
Act as guides for the Argonaute complex which cleaves the mRNA
Downregulates (decreases) expression

This mechanism is exploited for a technique called RNA interference

Question 21

How can we make a functional protein?

Answer 21

Need to fold the protein into correct 3D conformation

Covalent modifications – may need to be transported to the correct place for this to occur

Binding to other proteins, e.g. when assembling a complex

Question 22

How is protein digested by proteasome?

Answer 22

Proteins are marked for destruction by addition of ubiquitin molecules to lysines
Recognised by the proteosome complex
This degrades the protein into short peptides

Question 23

What is protein phosphorylation?

Answer 23

A phosphate group is transferred from ATP to an amino acid side chain of the protein by a protein kinase

Removal of a phosphate group is catalysed by a protein phosphatase

Phosphorylation often dictates whether a protein is in the ‘active’ state

Question 24

What is protein glycosylation? (1)

Answer 24

Attachment of carbohydrates to form glycoproteins
Does not use a template

Occurs in the Endoplasmic Reticulum and the Golgi Apparatus

Question 25

What is protein glycosylation? (2)

Answer 25

Promotes protein folding
Can limit binding by other proteins and increase resistance to digestion by proteases
Many important regulatory and functional roles

Question 26

What are the active and inactive forms of GTPase?

Answer 26

GTPases (e.g. Ras) cycle between active/inactive forms
The active form is bound to the nucleotide GTP
The inactive form is bound to GDP