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Genes and Bioinformatics > Week 4B - Control of Gene Expression in Eukaryotes - Part I > Flashcards

Week 4B - Control of Gene Expression in Eukaryotes - Part I Flashcards

1
Q

Eukaryotic gene expression is usually controlled at the level of

A

initiation of transcription
• by opening the chromatin

• local structure of the gene is changed –> general transcription apparatus binds to promoter

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2
Q

RNA is modified and processed

A

can control expression of alternative products from gene
• mRNA is exported from nucleus to cytoplasm
• mRNA is translated and degraded

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3
Q

Linker scanning mutagenesis can be used to

A

determine regulatory regions
• regulatory elements in promoters were identified by systematic replacement of short DNA segments with a DNA linker containing a random sequence of exactly the same size
• first used to search the promoter of the thymidine kinase gene is HSV
• microinjection into Xenopus oocytes allowed assaying of gene activity

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4
Q

Linker scanning

A

mutagenesis picture *

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5
Q

Thymidine kinase promoter elements identified by linker scanning

A

• overlapping linker scanning mutations were performed from one end of the region under investigation to the other
(each rectangle = a position in which a linker replaced a 6-10 nucleotide segment)
• experiment shows that thymidine kinase gene transcription is blocked by mutation in 3 distinct regions/sequence motifs that are just upstream from the transcription initiation site
• first is the TATA box (part of the core promoter for TBP), the CCAAT box, and the GC box

(ID regions in promoter responsible for transcription)

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6
Q

Experiment shows that thymidine kinase gene transcription is blocked by mutations in

A

3 distinct regions/sequence motifs that are just upstream from the transcription initiation site
• TATA box (part of the promoter for TBP)
• CCAAT box
• GC box

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7
Q

The proximal promoter provides

A

regulatory elements

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8
Q

The proximal promoter provides regulatory elements

A
  • CCAAT box and the GC box are present in the promoters of many eukaryotic genes between 50 and 200 bp upstream from the transcription initiation site
  • the region just upstream from the core promoter is called the proximal promoter
  • CCAAT and GC boxes are promoter proximal elements
  • there are many types of PPEs
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9
Q

The CCAAT box and the GC box are present in the promoters of many eukaryotic genes between

A

50 and 200 bp upstream from the transcription initiation site

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10
Q

Proximal promoter

A

the region just upstream from the core promoter

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11
Q

CCAAT and GC boxes are

A

promoter proximal elements (PPEs)

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12
Q

Core promoter binds

A

RNA polymerase

• machinery binds that transcribes

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13
Q

Regulatory promoters are often

A

cell/tissue specific

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14
Q

Enhancers

A

stimulate transcription

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15
Q

Silencers

A

block transcription

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16
Q

SV40 PPE consists of

A

6 GC boxes (GGGCGG)
• an additional regulatory sequence resides between 116-261 upstream of the transcriptional start site - enhancer
• enhancer regulatory sequences stimulate transcription

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17
Q

Enhancer…

A

an additional regulatory sequence resides between 116-261 upstream of the transcriptional start site
• enhancer regulatory sequences stimulate transcription

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18
Q

If you remove the enhancer

A

you still have expression but less

• put next to regulatory promoters = enhance transcription of core promoter

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19
Q

Properties of enhancers

A

• can act at a distance
• are orientation-dependent
• position can be upstream or downstream of the transcription initiation site
• can be cell type or tissue specific
(ie enhancer is active in 1 cell type or tissue type but NOT in another)
• a cell must have transcription activator proteins capable of binding to the enhancer for full gene expression to occur
• activator proteins an act in a number of different ways to turn on gene expression including directly interacting with the PIC and by promoting open chromatin structure (euchromatin)

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20
Q

Properties of silencers

A
  • eukaryotes also have negative regulatory elements called silencers which are sequence specific DNA elements that repress transcription of a target gene
  • mostly function independently of distance and orientation from/to target gene
  • binding sites for negative transcription factors - transcriptional repressors
  • these proteins act by establishing repressive chromatin (heterochromatin), prevent nearby transcriptional activator from binding to its binding site or by blocking PIC formation
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21
Q

Eukaryotes also have negative regulatory elements called

A

silencers

• sequence-specific DNA elements that repress transcription of a target gene

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22
Q

Silencers

A

sequence specific DNA elements that repress transcription of a target gene

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23
Q

Silencers are binding site for

A

negative transcription factors

• transcriptional repressors

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24
Q

General organization of control elements that regulate gene expression in higher eukaryotes

A
  • in addition to the core promoter (TATA box, Inr, DPE) which bind the basal transcriptional machinery, mammalian genes contain promoter proximal elements, enhancers, and silencers
  • many different enhancers have been characterized ranging in size from 50bp to 1.5 kbp
  • enhancers, silencers, and PPEs consist of clusters of modules (DNA sequence motifs) which bind specific transcriptional activator or transcriptional repressor proteins
  • this proves a mechanism for gene regulation by controlling the amount of functional transcription activator or repressor proteins with the cell nucleus
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25
Q

Core promoter binds

A

basal transcriptional machinery

TATA box, Inr, DPE = core promoters

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26
Q

Enhancers, silencers, and PPEs consist of clusters of modules (DNA sequence motifs) which bind

A

specific transcriptional activator or transcriptional repressor proteins

27
Q

Activator proteins interact with specific

A

promoter elements

28
Q

Activator proteins interact with specific promoter elements

A

• transcription activator proteins have at least 2 independently folded and distinct functional domains
– a DNA-binding domain makes sequence specific contacts with the control elements in the regulatory promoter or enhancer
– an activation domain is left “free” to recruit/bind various components of the transcription machinery or to alter chromatin structure around the transcriptional start site, in order to activate transcription
• additional domains include: dimerization domains and ligand binding domains

29
Q

Transcription activator domains have at least 2 independently folded and distinct functional domains
• DNA-binding domain

A

makes sequence specific contacts with the control elements in the regulatory promoter or enhancer

30
Q

Transcription activator domains have at least 2 independently folded and distinct functional domains
• activation domain

A

left free to recruit/bind various components of the transcription machinery
or to alter chromatin structure around the transcriptional start site
in order to activate transcription

31
Q

Electrophetic mobility shift assay (EMSA)

A
  • used to determine protein-DNA binding
  • label DNA with radioisotope and add protein or fractions of nuclear extract
  • electrophoretic mobility of DNA fragment I reduced (retarded) when complexed to proein, causing a shift in the location of the radiolabelled DNA fragment
  • visualized by autoradiography on X-ray film
32
Q

EMSA basic

A

• radiolabeled DNA + protein
• binds –> retardation/shift in DNA size
= fractioning, get which protein it is to get exact nucleotides

33
Q

DNase I footprinting assay

A
  • used to determine the exact position/sequence a protein binds to DNA
  • label DNA with radioisotope and add protein or fractions of nuclear extract
  • protein bound to DNA protects that region from digestion by a nuclease
  • region of DNA protected by the bound protein (and therefore the binding site) appears as a gap or footprint in the array of bands
  • visualized by autoradiography o X-ray film after electrophoresis
34
Q

DNase footprinting assay

A 
• DNA of known sequence
• nuclease --> ladder of sizes
• add sample with protein
• nuclease can't cut DNA where the protein is bound 
--> relate to sequence started with it
35
Q

A cell-based assay for transcription activation by an activator protein

A
  • transcriptional activators or repressors can be assayed for an ability to activate or repress transcription in an in vivo transfection assay
  • system requires 2 plasmids, one containing the putative transcriptional activator or repressor and the other contains a reporter gene and one or more binding sites for the protein
  • both plasmids are transfected into cells at the same time and the production of the reporter gene mRNA (and protein) is measured
  • the reporter gene often encodes green fluorescent protein for ease of assay
  • useful for using domain/truncated proteins to identify/map activator/repressor domains
  • plasmid 1 - clone gene you suspect is transcriptional activator/repressor (factor)
  • is the gene activating or repressing?
  • promoter sequence in plasmid 2 in front of promoter
  • both into cell, protein expressed and binds promoter affects transcription of reporter gene
36
Q

Use of deletion mutants identifies

A

functional domains in transcriptional activators

37
Q

Use of deletion mutants identifies functional domains in transcriptional activators

A
  • GAL4 is a yeast transcriptional activator which binds to UAS sequences
  • use of deletion mutants identified both the DNA binding domain (as 1-74) and the activation domain (as 738-823)
  • DNA binding regions are determined by EMSA and activation regions by cell-based reporter assay
  • what regions bind, what regions activate/repress
  • clone GAL4, map
  • 50 off start = lost DNA binding, receptor not expressed
  • deletion mapping proteins to find function
  • chop off end = bind but no activation
  • binding and activating domains separate
38
Q

picture for

A

cell-based assay for transcription activation by an activator protein *

39
Q

Modular structure of transcriptional activators

A
  • often contain more than one activation domain but usually only 1 DNA-binding domain
  • transcriptional activator proteins are often grouped according to the structure of their DNA-binding domains and can be classified into numerous structural types
  • more than 1 activation domain = activated by more than 1 signal
40
Q

Homeodomain proteins have

A

activation and DNA -binding domains
• homeobox proteins contain a DNA-binding region called the homeodomain
• the homeodomain contains a helix-turn-helix motif which is highly conserved between different homeodomain-containing proteins
• the recognition helix binds to the major groove of DNA and mediates sequence-specific binding
• regulate many important developmental genes

41
Q

Homeobox proteins contain a DNA_binding region called

A

the homeodomain

42
Q

The homeodomain contains a

A

helix-turn-helix motif which is highly conserved between different homeodomain-containing proteins

43
Q

The recognition helix binds

A

the major groove of DNA and mediates sequence specific binding

44
Q

Homeodomain proteins have

A

activation and DNA binding domains

• switches on very specific genes

45
Q

The zinc finger motif

A
  • many eukaryotic proteins have regions that fold around a central Zn2+ ion
  • a pair of cysteines and a pair of histidines act together to bind a zinc ion, causing a fold known as a zinc finger
  • this C2H2 zinc finger is the most common DNA-binding motif encoded in the human genome
  • 22- to 26- amino acid consensus
  • many proteins contain multiple C2H2 zinc fingers
  • 2 cysteine 2 histidine brought together, coordinated by zinc (cysteine and histidine are rare, so found together probably related)
  • fingers to probe DNA sequence
46
Q

3D structure of a zinc finger

A
  • the finger region consists of 2 antiparallel β sheets followed by an α-helix with the zinc ion buried in the interior
  • zinc ion binds with tetrahedral geometry to Cys-3 and Cys-6 in the β-strand and to His-19 and His-23 in the α helix
47
Q

The 3 Cys2His2 zincc fingers of Zif268 interact with adjacent 3 bp sites

A
  • 3 zinc finger protein interacts with 3bp sub-sites on DNA as the zinc-finger regions wraps around one turn of the DNA helix
  • overall binding specificity and affinity are determined by contributions from all 3 zinc fingers
  • zinc fingers have transcription factors
  • more fingers = recognize longer DNA (unique sequence)
48
Q

Nuclear receptors share a common domain structure

A
  • the nuclear receptor superfamily of transcriptional activators have a common structural design and contain an entirely different kind of zinc finger motif
  • highly conserved DNA-binding domain (DBD) ad conserved ligand-binding (hormone-binding domain)
  • activation function domains (AF1 and 2) and variable hinge region and are not conserved
  • nuclear receptors have ligand-binding domain + hinge + DNA binding domain
  • 2 transcriptional activator domains
  • signal - bind to DNA and start transcription
  • external signal –> gene response
49
Q

Hormone dependent gene activation by the nuclear receptor superfamily of zinc finger proteins

A
  • in the absence of hormone the receptor is kept in the cytoplasm through an interaction with inhibitors
  • in presence of hormone, diffuses through plasma membrane and binds to LBD releasing inhibitor - then translocates to the nucleus and binds as a dimer (homo- or hetero-) to response elements through its DBD
  • bind chemicals in ligand-binding domain
  • hormone internalized, recognized by ligand binding domain = transcription factor in, lose inhibitor –> into nucleus to start transcription
  • have response elements

hormone + DNA binding = affect transcription

50
Q

The DNA-binding domain of the GR receptor has 2 Cys4 zinc fingers

A
  • glucocorticoid receptor DVD has 2 Cys4 zinc fingers
  • no histidine (C4, not C2H2)
  • bind palindrome, bind 2 halves of DNA sites
51
Q

Glucocorticoid receptor binding domain bound to DNA

A
  • binds to DNA as a homodimer
  • contact bases in the major groove on the same side of the DNA molecule
  • binds to a DNA region known as the glucocorticoid repsonse element (GRE) consisting of two 6-bp palindromic half sites separated by 3 nucleotides
52
Q

Nuclear receptor domain swapping reveals function

A
  • effects of exchanging the DNA-binding domain of ER with that of GR
  • chimera induces expression of GR responsive genes in response to treatment with estrogen
53
Q

DNA-binding domains with leucine zippers

A

• identified in a number of proteins that contained a leucine every 7th amino acid over a 35 amino acid region
• predicted to form an α-helix in which Leu would occur every 2 turns
(α-helix has 3.6 amino acids per turn) - therefore Leu sidechains occur on 1 face
• proteins are characterized by homo- and hetero- dimerization through these α-helical segments
• Leu side chains interdigitate like teeth on a zipper

on α-helix get Leucine on same side if Leucine every 7 –> dimerize
• transcriptional diversity by binding together different monomers

54
Q

Leucine zippers form

A

a 2-stranded parallel coiled coil

55
Q

Leucine zippers form a 2-stranded parallel coiled coil

A
  • the leucine zipper does NOT directly bind to the DNA
  • it facilitates dimerization of the protein
  • the dimerization region often forms a coiled coil to join 2 proteins and facilitate subsequent DNA binding
  • the adjacent DNA-binding domain is highly basic and together with the leucine zipper forms the bzip DNA binding domain

• bring together 2 monomeric proteins

  • dimerize with leucine
  • 1 DNA binding domain brought together with another = bind DNA
56
Q

Gcn4 contains a bzip DNA binding domain

A
  • binds in major groove to 2 semi-palindromic half site

* homodimerization

57
Q

bZip heterodimerization

A
  • c-Jun can bind DNA as a homodimer or as a heterodimer with c-Fos
  • dimerization by the leucine zipper allows different complexes with different binding affinities and different activity to from on an identical DNA-binding site

Fos and Jun can make homo- or heterodimers
• related - both with leucine zipper and basic DNA binding region
• integrate different signals into 1 transcription

58
Q

Fos-Jun heterodimer bound to DNA

A
  • dimerization domain

* DNA-binding domain

59
Q

Basic helix-loop-helix (bHLH) domain

A
  • activators with bHLH domain bind to DNA as homo- or heterodimers
  • contain a basic recognition helix and HLH dimerization domain
60
Q

bHLHzip proteins have both

A

HLH and leucine zipper motifs
• Myc proteins for heterodimers with a related protein called Max
• Max can also form a heterodimer with another bHLH zip protein called Mad

• Myc dimerizes with related or unrelated protein

61
Q

Dimerization between factors provides

A

additional regulation and diversity

62
Q

Diversity in

A

regulation of transcription

63
Q

Proteins for specific

A

DNA sequence then bind genes so on/off

64
Q

Forming homo- or heterodimers integrates 2 different biological signals or bind 2 different DNA sites

A

2 different biological signals or bind 2 different DNA sites

Genes and Bioinformatics (10 decks)

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