Cells To Organisms Class > Lecture 4 > Flashcards

Lecture 4 Flashcards

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1
Q

Why are protein-coding genes more than just coding sequences?

A

They contain regulatory regions that control when, where, and how much a gene is expressed.

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2
Q

What are exons?

A

Regions of a gene that encode the protein.

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3
Q

What are introns?

A

Non-coding regions that are removed during RNA splicing.

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4
Q

Where can regulatory regions be located in a gene?

A

Upstream, downstream, or within the gene itself.

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5
Q

What are cis-regulatory elements?

A

DNA regions that control gene expression.

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6
Q

What information do cis-regulatory elements encode?

A

Where, when, how, and at what level a gene is expressed.

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7
Q

Q: Name examples of cis-regulatory elements.

A

Promoters, enhancers, and silencers.

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8
Q

Q: What are trans-acting factors?

A

Usually proteins that bind cis-regulatory elements to regulate transcription.

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9
Q

Q: What are transcription factors?

A

Trans-acting factors that bind DNA directly or associate with DNA-bound protein complexes.

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10
Q

What enzyme is required to transcribe protein-coding genes?

A

RNA polymerase II.

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11
Q

What are 3 examples of motifs?

A

Helix - turn - helix, leucine zipper, zinc finger motif

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12
Q

Why does RNA polymerase II require general transcription factors?

A

It cannot bind promoters or initiate transcription on its own.

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13
Q

Do all general transcription factors bind DNA directly?

A

No, many bind indirectly via protein–protein interactions.

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14
Q

What is the TATA box?

A

A promoter DNA sequence located ~30 bp upstream of the transcription start site.

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15
Q

Why is it called the TATA box?

A

It contains the conserved DNA sequence T-A-T-A.

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16
Q

Which protein recognises the TATA box?

A

TBP (TATA-binding protein).

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17
Q

What drives transcription?

A

Load up general transcription factor so then brings in RNA polymerase and it drives transcription

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18
Q

What groove does the TATA protein bind to? major or minor

A

minor

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19
Q

What is beneficial about the TATA box binding to the minor groove

A

it allows the DNA to more easily bend / melt for transcription and binding of RNA polymerase

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20
Q

What structural change occurs when TBP binds the TATA box?

A

The DNA bends ~80°.

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21
Q

Why are T-A base pairs easier to bend than G-C pairs?

A

They have fewer hydrogen bonds.

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22
Q

What is the main role of general transcription factors?

A

To help RNA polymerase II initiate transcription.

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23
Q

How do general transcription factors position RNA polymerase II?

A

By assembling at the promoter and aligning it correctly.

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24
Q

What role do they play in DNA unwinding?

A

They help separate DNA strands to expose the template.

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25
Why are they called “general” transcription factors?
They are required at nearly all RNA polymerase II promoters.
26
What naming system is used for general transcription factors?
TFIIA, TFIIB, TFIID, etc. (TFII = transcription factor for polymerase II)
27
Which general transcription factor contains TBP?
TFIID.
28
How are general transcription factors similar to bacterial sigma factors?
They perform equivalent promoter-recognition functions
29
What is the order of TFIID and TBP for binding to the promoter?
TFIID via TBP.
30
What happens after TBP binds the TATA box?
DNA bends, allowing other transcription factors to bind.
31
What is the role of TFIIH?
Uses ATP to unwind DNA, expose TEMPLATE strand and phosphorylate RNA polymerase II.
32
What does phosphorylation of RNA polymerase II cause?
transcription elongation and release of general transcription factors
33
Where do specific transcription factors usually bind?
Enhancer and silencer regions but can bind closer to the promotor.
34
What do enhancers do?
Increase transcription when bound by activator proteins.
35
What do silencers do
Decrease transcription when bound by repressors.
36
What do specific transcription factors influence to initiate transcription?
By influencing the transcription initiation complex.
37
How long are typical transcription factor binding motifs?
6–12 base pairs.
38
where motif do transcription factors recognise to bind to?
binding motif
39
Why are short binding motifs problematic?
They are more likely to occur randomly in the genome.
40
What does letter height represent in a sequence logo?
Frequency of that base at that position.
41
What do stacked letters in a sequence logo indicate?
Variability at that position.
42
what is the DBD, where does it bind?
DNA binding domain and it binds to the DNA
43
what is a TAD and what does it do?
Transactivation domain and it's not apart of the DNA, its required to activate other things for transcription
44
How can enhancers influence promoters thousands of base pairs away?
By looping DNA to bring them into proximity with the promotor.
45
what do the enhancers and silencers interact with?
Transcription initiation complex via the mediator complex
46
What complex connects enhancers to the transcription machinery?
The Mediator complex.
47
Are mediator complexes the same in different cells
No, they are different depending on the cell
48
Why do different cells express different genes?
They express different specific transcription factors.
49
What is a gene regulatory hierarchy
When transcription factors regulate other transcription factors.
50
What happens to oxygen deeper in organisms
Oxygen becomes limited deeper within tissues.
51
What is hypoxia?
A condition of low oxygen availability.
52
Why is hypoxia sensing important?
It stimulates blood vessel growth to improve oxygen delivery.
53
What is HIF1A?
A hypoxia-inducible transcription factor.
54
What does proline hydroxylase (from oxygen) do to HIF - 1a
Hydroxylates the proline found in HIF-1a
55
What does the hydroxlyation modification allow?
The modification is recognised by pVHL
56
Which protein recognises modified HIF1A?
pVHL.
57
What happens after pHVL is recognises modified HIF-1a and what does it result in?
Poly-ubiquitylation occurs and then is degraded in proteasome
58
Why does HIF1A remain stable under hypoxic conditions?
Proline hydroxylase requires oxygen and cannot modify HIF1A.
59
what happens to HIF-1a during hypoxia?
HIF -1a and HIF -1b bind to a VEGF Gene and can lead to transcription
60
What genes does stable HIF1A activate?
Genes such as VEGF involved in blood vessel growth.
61
What is the central dogma of molecular biology?
DNA → RNA → Protein.
62
How can we discuss Gene expression?
In terms of protein made or amount of mRNA made
63
Why measure both mRNA and protein levels?
They do not always correlate.
64
RT-PCR: Why is mRNA converted to cDNA?
Most molecular techniques work on DNA, not RNA.
65
RT-PCR: What does cDNA represent?
Only expressed, spliced mRNA sequences.
66
what does the mRNA hybridize with
poly-T primer
67
Why does a poly-T primer only work for eukaryotic mRNA?
It binds the poly-A tail.
68
When are random hexamer primers used?
When RNA lacks a poly-A tail (e.g. prokaryotes).
69
What occurs once poly-t primer is bound
makes compliementary DNA copy with reverse transcriptase - cDNA and mRNA helix
70
what is used to remove mRNA from the cDNA/mRNA helix
use RNAse H, this degrades RNA
71
what does DNA polymerase do?
synthesize second cDNA strand - making a cDNA/cDNA helix
72
What happens during PCR denaturation?
DNA strands separate at ~94–98°C.
73
What happens during primer annealing?
Primers bind to complementary sequences at ~50–65°C.
74
What happens during extension?
DNA polymerase synthesises new DNA at ~72°C.
75
Why does PCR amplify DNA exponentially?
Each cycle doubles the DNA amount.
76
What is the purpose of a molecular weight ladder?
To confirm PCR product size.
77
why is dye needed to be used
cause the DNA to glow
78
Why is GAPDH used as a control?
It is a housekeeping gene with stable expression.
79
What does a “No RT” control test for?
Genomic DNA contamination.
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