Chapter 3 – Regulation of gene expression
Transcription
Transcription: DNA (m)RNA – takes place in nucleus
Translation: mRNA protein – takes place on ribosomes in the cytoplasm
Regulatory elements in promotor region
Promotor: site for initiation of transcription on 5’ side it contains:
1. TATA-box; docking site for RNA polymerase II (holo-enzyme)
2. Transcription factor response elements; specific
sequences of DNA recognized by transcription
factors
Transcription factor binds to response element RNA
polymerase II (holo-enzyme) binds to TATA-box
transcription starts
Transcription factors
± 3000 TF in humans
TF can bind to DNA
They recognize specific DNA sequence (response elements)
Co-activators / suppressors are supportive molecules that
interact with the DNA-binding proteins (TF) to promote /
suppress RNA transcription
TF activity regulation:
1. Dimerization: TF never acts by itself but requires another protein to dimerize
with
2. Ligand binding: TF requires ligands to bind with
3. Expression restricted to particular cell types: specific TF give cell their
identities
4. Covalent modifications (phosphorylation): some TF are phosphorylated and
cannot enter the nucleus or if they are not phosphorylated they can enter the
nucleus
5. Cellular localization
TF consists of number of domains with specific functions:
All TF:
DNA binding domain; enable transcription
factor to bind to DNA
o Zinc finger
o Helix-loop-helix
o Helix-turn-helix
o Leucine zipper
Transcriptional activation domain (connects TF to transcription machinery (Pol
II)
Some TF: (domains to control activity of TF)
Dimerization domain: facilitates protein-
protein interactions between the 2 molecules
, 3 TF can give rise to 6 different dimers with different transcriptional activation
properties. 3 homodimers and 3 heterodimers
o Example: AP-1 (activation protein 1); jun and fos genes
18 possible combinations are possible
Jun = pro-proliferative
Jun B = anti-proliferative
Ligand binding domain: some transcription factors only function upon binding
of a ligand
Example is steroid hormone receptors
o Example: retinoic acid (vitamin A) receptor (RAR); RAR repress
transcription in the absence of RA
RA interact with ligand binding domain
receptor and transcription takes place
o Example:
Glucocorticoid interact with ligand binding
domain. The ligand binding domain is
required for the glucocorticoid to move to
the nucleus and bind to response element
in DNA
All the domains function independently
How to examine (onderzoeken) TF binding?
1. EMSA (electrophoretic mobility shift assay): piece
of radiolabelled DNA is used
a. Put on a gel; band low in the gel
b. Nuclear extract is added to radiolabelled
DNA allow transcription factors to find a
response element put on a gel; band high in the gel
because migration is slowed down
2. DNAse fooprinting:
DNAse is used to chop of the DNA. If there is a protein (response
element)present on this DNA it will protect the DNA from being
degraded by DNAse.
This is put on a gel. On the left there are no proteins so a lot of
degradation on the right there are proteins so a lot of protection from
degradation
3. ChIP-seq experiment: allow you to get a genome
wide view of where your protein of interest is bound
to DNA
DNA + bound protein fragment DNA perform
immunoprecepitate; antibodies bind to your protein
of interest release DNA prepare sequencing
library sequence map sequence tags to
genome & identify peaks
How to examine promotors/enhancers?
Transcription
Transcription: DNA (m)RNA – takes place in nucleus
Translation: mRNA protein – takes place on ribosomes in the cytoplasm
Regulatory elements in promotor region
Promotor: site for initiation of transcription on 5’ side it contains:
1. TATA-box; docking site for RNA polymerase II (holo-enzyme)
2. Transcription factor response elements; specific
sequences of DNA recognized by transcription
factors
Transcription factor binds to response element RNA
polymerase II (holo-enzyme) binds to TATA-box
transcription starts
Transcription factors
± 3000 TF in humans
TF can bind to DNA
They recognize specific DNA sequence (response elements)
Co-activators / suppressors are supportive molecules that
interact with the DNA-binding proteins (TF) to promote /
suppress RNA transcription
TF activity regulation:
1. Dimerization: TF never acts by itself but requires another protein to dimerize
with
2. Ligand binding: TF requires ligands to bind with
3. Expression restricted to particular cell types: specific TF give cell their
identities
4. Covalent modifications (phosphorylation): some TF are phosphorylated and
cannot enter the nucleus or if they are not phosphorylated they can enter the
nucleus
5. Cellular localization
TF consists of number of domains with specific functions:
All TF:
DNA binding domain; enable transcription
factor to bind to DNA
o Zinc finger
o Helix-loop-helix
o Helix-turn-helix
o Leucine zipper
Transcriptional activation domain (connects TF to transcription machinery (Pol
II)
Some TF: (domains to control activity of TF)
Dimerization domain: facilitates protein-
protein interactions between the 2 molecules
, 3 TF can give rise to 6 different dimers with different transcriptional activation
properties. 3 homodimers and 3 heterodimers
o Example: AP-1 (activation protein 1); jun and fos genes
18 possible combinations are possible
Jun = pro-proliferative
Jun B = anti-proliferative
Ligand binding domain: some transcription factors only function upon binding
of a ligand
Example is steroid hormone receptors
o Example: retinoic acid (vitamin A) receptor (RAR); RAR repress
transcription in the absence of RA
RA interact with ligand binding domain
receptor and transcription takes place
o Example:
Glucocorticoid interact with ligand binding
domain. The ligand binding domain is
required for the glucocorticoid to move to
the nucleus and bind to response element
in DNA
All the domains function independently
How to examine (onderzoeken) TF binding?
1. EMSA (electrophoretic mobility shift assay): piece
of radiolabelled DNA is used
a. Put on a gel; band low in the gel
b. Nuclear extract is added to radiolabelled
DNA allow transcription factors to find a
response element put on a gel; band high in the gel
because migration is slowed down
2. DNAse fooprinting:
DNAse is used to chop of the DNA. If there is a protein (response
element)present on this DNA it will protect the DNA from being
degraded by DNAse.
This is put on a gel. On the left there are no proteins so a lot of
degradation on the right there are proteins so a lot of protection from
degradation
3. ChIP-seq experiment: allow you to get a genome
wide view of where your protein of interest is bound
to DNA
DNA + bound protein fragment DNA perform
immunoprecepitate; antibodies bind to your protein
of interest release DNA prepare sequencing
library sequence map sequence tags to
genome & identify peaks
How to examine promotors/enhancers?
