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Human Molecular B > Lecture 7 - Control Of Gene Expression > Flashcards

Lecture 7 - Control Of Gene Expression Flashcards

1
Q

At what level is Eukaryotic gene expression usually controlled?

A

Eukaryotic gene expression is usually controlled at the level of initation of transcription by affecting either formation of PIC or by regulating local chromatin structure

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2
Q

TATA box

A

TATAXAX consensus sequence(X - A or T) Located 25-30 bp upstream of the transcription start site

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3
Q

Initiator element (lnr)

A

Overlaps the transcription initiation site

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4
Q

Downstream promoter element

A

Extends from about +28 to +34

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5
Q

What is XCPE1?

A

Present in TATA-less human core promoters (1% human gene)

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6
Q

What method can determine regulatory regions?

A

Linker scanning mutagenesis

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7
Q

Linker scanning mutagenesis

A

Regulatory elements in promoters were identified by systematic replacement of short DNA segment with a DNA linker containing a random sequence of exactly the same size

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8
Q

When was linker scanning mutagenesis first used?

A

Search the promoter of the thymidine kinase gene in HSV

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9
Q

What did microibjecton into Xenopus ooocytes allow?

A

Assaying of gene activity (i.e. amount of MRNA produced)

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10
Q

Linker scanning for thymidine kinase

A

Overlapping linker scanning were performed from one end of the region under investigation to the other Experiments show that thymidine kinase gene transcription is blocked by mutations a in 3 distinct regions/sequence motifs that are just upstream from the transcription initiation site

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11
Q

What are the three distinct regions

A

TATA box (core promoter for TBP) CCAAT box GC box

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12
Q

CCAAT box

A

GGCCAATCT

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13
Q

GC box

A

GGGCGG

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14
Q

Where would you find the CCAAT and GC box?

A

Promoters of many eukaryotic genes between 50 and 200 bp upstream from the transcription initiation site

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15
Q

What is the region upstream of the core promoter called?

A

Proximal promoter

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16
Q

What are CCAAT and GC boxes?

A

Promoter proximal elements

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17
Q

What is SV40 PPE made up of?

A

6 GC boxes

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18
Q

Where would you find an additional regulatory sequence?

A

Residing between 116-261 upstream of the Transcriptional start site: enhancer

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19
Q

What is the enhancer role?

A

Stimulate transcription

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20
Q

What can silencers do?

A

Block transcription

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21
Q

What are the properties of enhances?

A

Can act at a distance Orientation independent Position can be upstream or downstream of transcription initiation site Cell type or tissue specific A cell must have transcription activator proteins capable of binding to enhancer for full gene expression to occur Activation proteins can act in a number of different ways to turn on gene expression including interacting with the PIC and by promoting open chromatin structure (Euchromatin)

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22
Q

What are the properties of silencers?

A

Eukaryotes also have negative regulatory elements that are sequence specific DNA elements that repress transcription of target gene Mostly function independently of distance and orientation from/to target gene Proteins act by establishing repressive chromatin, prevent nearby Transcriptional activators from binding to its binding site of blocking PIC formation

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23
Q

What are examples of core promoters?

A

TATA box Inr DPE

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24
Q

What do mammalian genes contain?

A

Promoter proximal element, enhancers and silencers

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25
Size of enhancers
Ranging in size from 50bp to 1.5kbp
26
What do enhancers, silencers and PPE consist of?
Cluster of modules (DNA sequence motif)
27
Where do Enhancers, silencers and PPE bind?
Specific Transcription activator or repressor proteins Mechanism for gene regulation by controlling the amount of functional transcription Activator or repressor proteins within cell nucleus
28
Transcription activator or repressor proteins have at least ... independently folded and distinct functional domains
2
29
What are the 2 functional domains?
DNA binding domain. Activation/repression domains
30
What is the DNA binding domain?
Make sequence specific contacts with the control elements in regulatory promoter or enhancer
31
Activation or repression domain
Left free to recruit or bind various components of transcription machinery or to alter chromatin structure around transcriptional start site in order to activate transcription
32
Additional functional domain that may exist
Dimerisation domain and ligand binding domain
33
EMSA
Electrophoretic mobility shift assay
34
The use of electrophoretic mobility shift assay
Determine protein-DNA binding
35
Method of EMSA
Label DNA with radioisotope and add protein or fractions of nuclear extrac. Electrophoretic mobility of DNA fragment reduced when complexed to protein Causes a shift in location of radiolabelled DNA fragment. Visualised by autoradiography on X-ray film
36
The use of the DNase I foot print assay
Determine the exact position/sequence a protein binds to DNA
37
Method of DNase I
Label DNA with radioisotope and add protein or fraction of nuclear extract Protein bound to DNA protects that region from digestion by nuclease Region of DNA protected by bound proteins appears as a GAP or footprints in array of bands Visualised by autoradiography in X-ray film after electrophoresis
38
Use of a cell based assay for transcription activation by an activator protein
Determine the type of activity of a DNA-binding protein that regulates transcription. Useful for using domains/truncated proteins to identify/map activator/repressor domains
39
Method of cell based assay for transcription activation by activator proteins
Transcriptional activators or repressors can be assuaged for an ability to activate or repress transcription in an in vivo transfection assay System requires 2 plasmids, one contains g the putative transcriptional activator or repressor and the other contains a reporter gene and one of more binding sites for protein Both plasmids are transfected into cells at the same time and the production of reporter gene MRNA and protein is measured The reporter gene often encodes green fluorescent proteins for ease of assay
40
What is the use of deletion mutants identifies functional domain in transcriptional activators
To determine and identify the functional domain in a DNA binding protein eg. An activation/repressor domain and the DNA-binding protein
41
What is GAL4?
A yeast transcriptional activator whin binds to UAS sequences
42
What does the use of deletion mutations identify?
Identified both DNA binding domain(aa 1-74) and activation domains (aa 738-823)
43
What determines DNA binding regions ?
EMSA
44
What determines DNA activation region
Cell based reporter assay
45
What is the molecular structure of transcriptional activators?
Often contain more than one activation domain but usually only one DNA-binding domain
46
How are transcriptional activators grouped?
Grouped according to the structures of their DNA-binding domains and can be classified into numerous structural types
47
Homeodomain
Homeobox proteins contain DNA-binding region Regulate many important developmental genes
48
What is the structure of homeodomain?
Helix-turn-helix motif which is highly conserved between different homeodomain-containing proteins
49
What is the role of recognition helix ?
Bind in the major groove of DNA and mediates sequence-specific binding
50
The zinc finger motif
Many eukaryotic proteins have region that fold around a central Zn2+ Ion A pair of cysteine and a pair of histidine act together to bind a zinc ion, cashing a fold know. As a zinc finger
51
Which zinc finger is the most common DNA-binding motif encoded I. The human genome?
C2H2 zinc finger
52
What is the size of C2H2 zinc finger?
23- to 26- amino acid consensus
53
Where is the C2H2 zinc finger found ?
Many proteins contain multiple C2H2 zinc fingers
54
What is the structure of C2H2 zinc finger?
The finger region consist of 2 anti-parallel B sheets followed by alpha helix with the zinc ion buried in the interior
55
How does Zinc ion bind?
tetrahedral geometry to cys-3 and cud-6 in Beta strand and to His-19 and His-23 in the alpha helix
56
What does the 3 Cys2His2 zinc fingers interact with?
Adjacent 3bp sites 3 zinc finger protein interacts with 3 bp sub-sites on DNA as the zinc finger regions wrap around s one turn of the DNA helix
57
What does overall binding specificity and affinity determined by?
Contributions from all three zinc fingers
58
In the absence of hormone what happens to hormone dependent gene activation by nuclear receptor super family?
Receptor is kept in the cytoplasm through an interaction with inhibitors
59
In the presence of hormone what happens to hormone dependent gene activation by nuclear receptor superfamily?
Hormone diffuses through the plasma membrane and binds to LBs releasing inhibitor translocates to the nucleus and bind as dimer to response element through zinc finger DNA binding domain
60
What do nuclear receptors share?
A common domain structure
61
Nuclear receptors
Highly conserved DNA-binding domain (DBD) and conserved ligand binding domain. Activation function domain and variable hinge region and are not conserved
62
Nuclear receptor superfamily
The nuclear receptor superfamily of transcriptional activators Have a common structural design and contain an entirely different kind of zinc finger motif
63
What is the effect of exchanging DNA binding domain of ER with GR?
Chimera induces expression of GR responsive genes in response to treatment with estrogen
64
What is the structure of DNA binding domain of the GR receptor ?
The DNA binding domain of the GR receptor has 2 Cys4 zinc fingers
65
Glucocorticoid receptor binding domain bound to DNA ?
Binds to DNA as homodimer Contact bases in the major groove on same side of the DNA molecule Bind to DNA region: Glucocorticoid response element consists of two 6-no palindromic half sites separates by 3 nucleotides
66
What does Gcn4 contain ?
bzip DNA binding domain
67
Where does a Gcn4 bind?
Binds in major groove to two semi-palindromic half sites
68
bZip heterodimerization?
Dimerisation by leucine zipper allows different complexes with different affinities and different activity to form on an identical DNA-binding site
69
What and how does c jun bind with DNA?
c-Fos As a homodimer or heterodimer
70
Basic helix loop helix
Activators with the bHLH domain bind to DNA as homo or heterodimers Contain a basic recognition helix and HLH dimerisation domain ?
71
What motifs do bHLHzip proteins have?
HLH and leucine zipper motifs
72
Myc heterodimers
Myc proteins for heterodimers with a related protein called Max
73
Max heterodimer
Max can also form a heterodimer with another bHLH zip protein called Mad
74
What does dimerisation between factors provide?
Additional regulation and diversity

Human Molecular B (13 decks)

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