Control of gene expression in eukaryotes: part 1
Learning outcome:
1. DNA regulatory elements
2. Regulatory proteins, domain organisation and DNA-binding structures
Control of gene expression
Eukaryotic gene expression is usually controlled at the level of initiation of
transcription by opening the chromatin.
DNA exists in a structure called chromatin, which means inaccessible to
transcription as packaged tightly.
Therefore, many processes regulate chromatin structure.
Prokaryotic DNA is circular and not packaged in a nucleus.
There are post-transcriptional ways of translating gene expression, as
mRNA needs to leave the nucleus and enter the cytoplasm for translation.
Regulation of gene expression - whether an mRNA molecule gets translated
into a protein.
Regulating gene expression posttranslational – is modifying mRNA to
whether it becomes a protein or not.
Linker scanning mutagenesis can be used to determine regulatory regions
Regulatory elements in promotors were identified by schematic replacement of short DNA segments with a DNA linker
containing a random sequence exactly the same size.
- DNA Linker= double stranded DNA in between 2 nucleosome cores that in association with histone H1, holds the
core together.
First used to search for promoter of the thymidine kinase gene in HSV.
Microinjection into xenopus oocytes allowed assaying of gene activity (i.e.
amount of mRNA produced)
- Assay = determine the content or quality
,Thymidine kinase promotor elements identified by linker scanning
150bp of promoter DNA from mammalian
TATA box is centred ~-20 to -30 upstream of start site
Clone promoter fragment then assay if there is RNA synthesis
- i.e. transcription of reporter gene (gene that researchers attach to a regulatory sequence of another gene of
interest)
- can determine where the important functional sequences are in DNA
Overlapping linker scanning mutations (rectangles) were performed from one end of the region under investigation to
the other
Each rectangle represents a position which a linker replaces a 6-10 nucleotide segment
If mutate the 2 rectangles corresponding to the TATA box, TFllD does not recognise the DNA sequence anymore and
does not recruit RNA polymerase ll and reporter gene is not expressed= no mRNA
Therefore, in the wild type DNA fragment there must be a DNA sequence element that affects transcription
Took the 150 - 200 bp fragment of genes and made a series of overlapping mutations
Not only the absence of TATA BOX result in loss of transcription of reporter gene
There was a number of other regions, which they identified were called the GC rich region and CAT boxes
The red mutations correspond to the GC and CCAAT box and resulted in no gene expression even in the presence of the
TATA box.
These are additional regulatory elements within promoters that have nothing to do with the TATA box that still resulted
in the absence of reduction in gene expression
The GC and CCAAT boxes are regulatory elements or promoters and upstream the core promoter
GC and CCAAT boxes are distinct from the TATA box, Inr, TBP binding elements, downstream promoter elements
The core promoter is required for the assembly of the preinitiation complex with RNA polymerase ll
From the mutagenesis studies, the preinitiation complex is not sufficient for robust transcription because when you do
linker scanning mutagenesis, can identify additional sequences within wider promoter region (which is called a
regulatory promoter) that also stop transcription happening even if the PIC is present
The GC and CCAAT boxes are present in many eukaryotic genes and they exert effect on transcription by recruiting
proteins that bind specifically to the DNA sequences
Experiment show that thymidine kinase gene transcription is blocked by mutation in 3 distinct regions/sequences
motifs that are upstream from the transcription initiation site.
First is the TATA box (part of the core promoter for TBP), the CCAAT box and GC box
, The proximal promoter provides regulatory elements
CCAAT box (GGCCAATCT) and the GC box (GGGCGG) are present in the promotor of many eukaryotic genes between 50
and 2002 bp upstream from the transcriptional initiation site.
Region upstream from the core promoter is called the proximal element.
CCAAT and GC boxes are promoter proximal elements
There are many other types of PPEs.
Enhancers stimulate transcription and silencers block transcription
SV40 is a virus
SV40 PPE consists of 6 GC boxes (GGGCGG)
an additional regulatory sequence residues between 116-261 upstream of the transcriptional start site : enhancers
Enhancer regulatory sequences stimulate transcription.
Using SV40 isolated a different kind of regulatory element
In the virus transcription takes place left and right ward and the control element is in the middle
Mutation in the control region allowed for identification enhancers because did not stop transcription rather enhancers
enhance transcription from the start site.
Enhancers are crucial to gene regulation
Properties of enhancers
Learning outcome:
1. DNA regulatory elements
2. Regulatory proteins, domain organisation and DNA-binding structures
Control of gene expression
Eukaryotic gene expression is usually controlled at the level of initiation of
transcription by opening the chromatin.
DNA exists in a structure called chromatin, which means inaccessible to
transcription as packaged tightly.
Therefore, many processes regulate chromatin structure.
Prokaryotic DNA is circular and not packaged in a nucleus.
There are post-transcriptional ways of translating gene expression, as
mRNA needs to leave the nucleus and enter the cytoplasm for translation.
Regulation of gene expression - whether an mRNA molecule gets translated
into a protein.
Regulating gene expression posttranslational – is modifying mRNA to
whether it becomes a protein or not.
Linker scanning mutagenesis can be used to determine regulatory regions
Regulatory elements in promotors were identified by schematic replacement of short DNA segments with a DNA linker
containing a random sequence exactly the same size.
- DNA Linker= double stranded DNA in between 2 nucleosome cores that in association with histone H1, holds the
core together.
First used to search for promoter of the thymidine kinase gene in HSV.
Microinjection into xenopus oocytes allowed assaying of gene activity (i.e.
amount of mRNA produced)
- Assay = determine the content or quality
,Thymidine kinase promotor elements identified by linker scanning
150bp of promoter DNA from mammalian
TATA box is centred ~-20 to -30 upstream of start site
Clone promoter fragment then assay if there is RNA synthesis
- i.e. transcription of reporter gene (gene that researchers attach to a regulatory sequence of another gene of
interest)
- can determine where the important functional sequences are in DNA
Overlapping linker scanning mutations (rectangles) were performed from one end of the region under investigation to
the other
Each rectangle represents a position which a linker replaces a 6-10 nucleotide segment
If mutate the 2 rectangles corresponding to the TATA box, TFllD does not recognise the DNA sequence anymore and
does not recruit RNA polymerase ll and reporter gene is not expressed= no mRNA
Therefore, in the wild type DNA fragment there must be a DNA sequence element that affects transcription
Took the 150 - 200 bp fragment of genes and made a series of overlapping mutations
Not only the absence of TATA BOX result in loss of transcription of reporter gene
There was a number of other regions, which they identified were called the GC rich region and CAT boxes
The red mutations correspond to the GC and CCAAT box and resulted in no gene expression even in the presence of the
TATA box.
These are additional regulatory elements within promoters that have nothing to do with the TATA box that still resulted
in the absence of reduction in gene expression
The GC and CCAAT boxes are regulatory elements or promoters and upstream the core promoter
GC and CCAAT boxes are distinct from the TATA box, Inr, TBP binding elements, downstream promoter elements
The core promoter is required for the assembly of the preinitiation complex with RNA polymerase ll
From the mutagenesis studies, the preinitiation complex is not sufficient for robust transcription because when you do
linker scanning mutagenesis, can identify additional sequences within wider promoter region (which is called a
regulatory promoter) that also stop transcription happening even if the PIC is present
The GC and CCAAT boxes are present in many eukaryotic genes and they exert effect on transcription by recruiting
proteins that bind specifically to the DNA sequences
Experiment show that thymidine kinase gene transcription is blocked by mutation in 3 distinct regions/sequences
motifs that are upstream from the transcription initiation site.
First is the TATA box (part of the core promoter for TBP), the CCAAT box and GC box
, The proximal promoter provides regulatory elements
CCAAT box (GGCCAATCT) and the GC box (GGGCGG) are present in the promotor of many eukaryotic genes between 50
and 2002 bp upstream from the transcriptional initiation site.
Region upstream from the core promoter is called the proximal element.
CCAAT and GC boxes are promoter proximal elements
There are many other types of PPEs.
Enhancers stimulate transcription and silencers block transcription
SV40 is a virus
SV40 PPE consists of 6 GC boxes (GGGCGG)
an additional regulatory sequence residues between 116-261 upstream of the transcriptional start site : enhancers
Enhancer regulatory sequences stimulate transcription.
Using SV40 isolated a different kind of regulatory element
In the virus transcription takes place left and right ward and the control element is in the middle
Mutation in the control region allowed for identification enhancers because did not stop transcription rather enhancers
enhance transcription from the start site.
Enhancers are crucial to gene regulation
Properties of enhancers
