Bio 3000 - Post Transcriptional Control of Gene Expression.
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How is Gene Expression controlled after Transcription?
- Altering mRNA stability.
- Altering Translational ability.
What is Iron Homeostasis?
Regulation of iron levels in cells (High Iron is Toxic, Low Iron is Anemic).
What is Transferrin?
A protein that binds extracellular Iron and is recognized by Transferrin Receptors.
How does Transferrin enter the cell?
- Through Vesicles.
- Transferrin Binds to Transferrin Receptors and then enters the cell in a Vesicle.
What is Ferritin?
A Molcule responsible for dealing with excess intracellular iron.
How do cells control intracellular iron?
By balancing the Ferritin to Transferrin Receptor Ratios.
What happens to Transferrin receptor (TfR) mRNA when iron levels drop?
What happens to Transferrin Receptor (TfR) mRNA when iron levels rise?
Which part of the TfR mRNA is responsible for responding to the changes in Iron concentration?
The 3' Untranslated Region.
How did researchers determine which part of the TfR mRNA is responsible for responding to changes in Iron Concentration?
- 1. Cells were transfected with plasmids missing different regions of the TfR mRNA.
- 2. They added a chelating agent to remove Cellular Iron.
- 3. Cells with the plasmid with a stunted 3' UTR showed no response to changes in iron concentration.
What is a Chelating agent?
A substance that binds to Iron.
What is unique about the 3' UTR in TfR mRNA?
It is unusually long.
What are Iron Response Elements (IREs)
Conserved Stem Loop elements whithin the 3' UTR of TfR mRNA and 5' mRNA coding Ferritin.
What are some characteristic features of IREs?
- Stem Loops (5).
- Conserved Loop "head" elements.
- Bulged C on the stem.
What is the sequence of the conserved loop on the IREs?
5' - CAGUG -3'
How many IRE's does Transferrin Receptor mRNA have?
How many IRE's does the 5' Ferritin mRNA have?
How did researchers find out which stem loops were responsible for iron responses?
They selectively removed IREs & observed the effect on responses to changing Iron.
What was the TRS-1 sequence element?
The TfR 3' UTR was missing the 5' & 3' (Outside) stem loop elements.
what was the TRS-3 sequence element?
The 3'-UTR with no IREs.
What is the TRS-4 sequence element?
The 3' UTR with all stem loops but no bulged C.
What was observed with protein output with the TRS-1, TRS-3 and TRS-4 sequence element?
- TRS-1: Normal response to changing iron.
- TRS-3: No Response to changing Iron (Protein produced).
- TRS-4: No response to changing iron, no protein produced.
What were the conclusions of the IRE experiment?
- 1. Some IREs are needed (TRS-1)
- 2. mRNA without IREs is more stable.
- 3. The IRE sequence mutation causes mRNA instability in all conditions.
What did the IRE experiment measure to quantify IRE effectiveness?
How do IRE's stabilize the TfR mRNA?
- The IRE's contain a bulged C which allows for binding of Aconitase in low iron concentrations.
- This binding prevents RNAase activity.
What is Aconitase?
A protein that binds to the IRE bulged C's & prevents cleavage by blocking the site.
What happens to IRE's in high Iron concentrations?
- 1. Iron binds to Aconitase.
- 2. Aconitase Dissociates
- 3. RNAase cleaves the 3' UTR.
- 4. Unstable mRNA is degraded.
Why is the TfR mRNA more stable when IRE's are removed?
- The 3' UTR will fold into an unusual secondary structure.
- This structure "hides" the RNAse cleavage site and prevents cleavage of the 3' UTR.
How do you perform a Northern Blot?
- Hybridize a labelled probe to RNA.
Bio 3000 - What is the purpose of a Northern Blot?
To visualize size & amount of mRNA product.
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