Transcript in the promoter?

NUTRITION AND GENE EXPRESSION
February 19, 2016
PROMOTER MUTATIONS
Mutations in the REGULATORY part of the gene
(usually called the PROMOTER) show us the
importance of those DNA sequences for the
control of gene activity.
To study biochemistry, we need tools to study
these complex pathways. The data from this study
illustrates the effects of promoter mutations, and
shows useful methods to study this question.
CASE STUDY
A mutation that led to low levels of human
growth hormone (HGH), and impaired growth.
How was the key mutation found, that caused
this disorder? In this case, there were low
levels of active growth hormone in the
bloodstream.
What DIFFERENT mutations might lead to low
levels of circulating HGH?
These findings are taken from:
Decreased Expression of the GHRH Receptor
Gene Due to a Mutation in a Pit-1 Binding Site
ROBERTO SALVATORI, XIAOGUANG FAN, PRIMUS E.
MULLIS, AZEB HAILE, AND MICHAEL A. LEVINE
Molecular Endocinrology, Vol 16: 450-458, 2002
BRIEF
SUMMARY:
Gene Expression
in Eukaryotes
This very typical
diagram DOES NOT
SHOW THE CONTROL
REGION OF THE GENE,
WHICH LEADS TO
CONFUSION!
GHRH
PITUITARY CELL
GH
Growth hormone
is released
When growth-hormone-releasing-hormone (GHRH)
binds to the receptor on the pituitary cell membrane,
growth hormone (GH) is released from a vesicle.
If the releasing receptor is absent or defective,
GH release can be impaired. This paper examined the
gene for the RECEPTOR.
The Growth-Hormone-Releasing-Hormone Receptor gene is on
chromosome 7 (at location 7p14), as shown in the diagram.
The investigators already found that there was a defect in the gene
on one chromosome. In the protein made by that gene, there was a
glutamic acid (negative charge) instead of lysine (positive charge) at
AA position 329, so the receptor was made, but did not work.
So what was the defect on the other chromosome?
Location of gene for the receptor
THE PROMOTER FOR THE GENE
FOR THE HUMAN GROWTH
HORMONE RELEASING RECEPTOR:
it’s about 2600 nucleotides
long. The receptor is located
on the surface of pituitary cells.
It responds to a 44-aa peptide
called SOMATOCRININ or HGRH.
If this receptor is not present and
functional, HGH is not released, and
very short stature is the result.
The transcription factor Pit-1
binds to the promoter which controls
expression of this receptor
at the sequence: TATGCAA.
Sites in RED bind Pit-1, which
is a TF with multiple binding
sites on the promoter.
REMEMBER: The gene includes
the promoter (which regulates
expression of the gene) and the
sequencewhich codes for the
protein itself.
It’s been shown that if promoter is
normal between –400 and –20, then
good expression of the gene will occur.
Therefore, investigators looked at the
promoter sequence near to the start site
for transcription of the receptor gene.
NORMAL Pit-1 site: TATGCAA
MUTANT Pit-1 site: TATGCCA
What did they find? In a child with short stature, they found a key
mutation in the PROMOTER for the gene that makes the growth
hormone releasing receptor, at position about (-120) from the
transcription start site. The change was from an AT base pair to
a CG base pair, in the DNA sequence in the promoter,
Therefore, this child might not be able to release normal
levels of HGH to the bloodstream. How can this be tested?
How we assess the SIGNIFICANCE of that mutation from
AC at ( –124) in the promoter?
For that purpose, we need to develop many TOOLS
to examine how gene expression works.
For example: if a person does not release enough HGH,
there might be MANY variations in the gene,
but majority of those variations are harmless.
We need strategies to determine which mutations
are really important!
If you have transcription factor bound to
the promoter, any gene that is downstream
will be transcribed. This is the most
important research technique to study
mechanisms of gene expression.
ALL THESE GENES (PROTEINS X, Y, AND Z)
HAVE THE SAME REGULATORY DOMAIN ARTIFICIALLY
ATACHED UPSTREAM OF THE CODING REGION
Protein X DNA
RNA for X
Protein Y DNA
RNA for Y
Protein Z DNA
RNA for Z
ATTCG
ATTCG
ATTCG
WITH TRANSCRIPTION FACTOR BOUND
UPSTREAM, RNA POL-II WILL MAKE
primary RNA transcript FOR ANY DOWNSTREAM DNA
LUCIFERASE CONSTRUCTS
Small, artificial genes that can be
placed inside cells. In the cell, the
gene can be activated and make
mRNA for luciferase, which is then
translated by the ribosomes.
The gene has to have a PROMOTER
and CODING REGION.
Requires the
enzyme
LUCIFERASE
Measured as a
burst of light!
TOTAL LIGHT GENERATED
ABUNDANT
LUCIFERASE
LIMITED AMOUNT
OF LUCIFERASE
ADD ATP
LUCIFERASE VECTORS: to examine if a TF in a
cell activates transcription from a promoter
Promoters, with different
DNA sequences
Luc coding domain: will make
mRNA for Luc, if transcribed.
The mRNA then makes the
Luc protein, which is measured
ORI
We compare both TATGCAA and mutant TATGCCA
in the promoter.
Promoters, with different
DNA sequences: Pit-1 might bind here
Luc coding domain: will make
mRNA for Luc, if transcribed
ORI
PLACE THIS CONSTRUCT
IN CELLS THAT MAKE
THE PIT-1 PROTEIN
TOTAL LIGHT GENERATED
NORMAL
PROMOTER:
TATGCAA
MUTANT
PROMOTER:
TATGCCA
CELL
EXRACTS
ADD ATP
These LUCIFERASE-containing artificial genes
were placed inside cells that made the Pit-1
transcription factor.
But if there were MUTATIONS in the promoter,
very small amounts of luciferase were made.
The Pit-1 transcription factor was not able to
properly bind to the mutant promoter.
LUCIFERASE
EXPESSION
No promoter
in vector
TATGCAA TATGCCA
in promoter in promoter
Mutation
elsewhere
in promoter
CONCLUSION:
This construct, containing the Luc gene, was placed
inside cells that contained the Pit-1 transcription factor.
HOWEVER: a small single base-substitution in the
promoter stopped Pit-1 from binding, and Luc mRNA
was not transcribed from the construct. Correspondingly,
the cells made much less luciferase.
THIS TECHNIQUE ALLOWED THE INVESTIGATORS
TO FIND THE CRITICAL MUTATION THAT CAUSED
LOW LEVELS OF HGH TO BE RELEASED.
Most of what we know about transcription factor interactions
with promoters comes from studies like this with
“artificial” chromosomes, with variations in the promoter.