nutrition and gene expression february 19, 2016 promoter mutations mutations in the regulatory part...
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NUTRITION AND GENE EXPRESSION February 19, 2016
PROMOTER MUTATIONS
Mutations in the REGULATORY part of the gene(usually called the PROMOTER) show us theimportance of those DNA sequences for thecontrol of gene activity.
To study biochemistry, we need tools to studythese complex pathways. The data from this studyillustrates the effects of promoter mutations, andshows 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
BRIEFSUMMARY:
Gene Expressionin Eukaryotes
This very typicaldiagram DOES NOTSHOW THE CONTROLREGION OF THE GENE,WHICH LEADS TOCONFUSION!
GH
Y
GHRH
Growth hormoneis 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 thegene for the RECEPTOR.
PITUITARY CELL
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 GENEFOR THE HUMAN GROWTHHORMONE RELEASING RECEPTOR:it’s about 2600 nucleotideslong. The receptor is locatedon the surface of pituitary cells.It responds to a 44-aa peptidecalled SOMATOCRININ or HGRH.
If this receptor is not present andfunctional, HGH is not released, andvery short stature is the result.
The transcription factor Pit-1binds to the promoter which controlsexpression of this receptor at the sequence: TATGCAA.
Sites in RED bind Pit-1, whichis a TF with multiple bindingsites on the promoter.
It’s been shown that if promoter is normal between –400 and –20, thengood expression of the gene will occur.
Therefore, investigators looked at the promoter sequence near to the start sitefor transcription of the receptor gene.
REMEMBER: The gene includesthe promoter (which regulatesexpression of the gene) and the sequencewhich codes for the protein itself.
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 toa CG base pair, in the DNA sequence in the promoter,
Therefore, this child might not be able to release normallevels of HGH to the bloodstream. How can this be tested?
How we assess the SIGNIFICANCE of that mutation from AC at ( –124) in the promoter?
For that purpose, we need to develop many TOOLSto 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 mutationsare 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
ATTCG
ATTCG
ATTCG
Protein X DNA
Protein Y DNA
Protein Z DNA
RNA for X
RNA for Y
RNA for Z
WITH TRANSCRIPTION FACTOR BOUNDUPSTREAM, RNA POL-II WILL MAKE
primary RNA transcript FOR ANY DOWNSTREAM DNA
LUCIFERASE CONSTRUCTS
Small, artificial genes that can beplaced inside cells. In the cell, thegene can be activated and makemRNA for luciferase, which is thentranslated by the ribosomes.
The gene has to have a PROMOTERand CODING REGION.
Requires theenzyme
LUCIFERASE
Measured as a burst of light!
TO
TA
L L
IGH
T G
EN
ER
AT
ED
ADD ATP
LIMITED AMOUNTOF LUCIFERASE
ABUNDANTLUCIFERASE
LUCIFERASE VECTORS: to examine if a TF in a cell activates transcription from a promoter
ORI
Luc coding domain: will makemRNA for Luc, if transcribed.The mRNA then makes theLuc protein, which is measured
Promoters, with differentDNA sequences
We compare both TATGCAA and mutant TATGCCA in the promoter.
ORI
Luc coding domain: will makemRNA for Luc, if transcribed
Promoters, with differentDNA sequences: Pit-1 might bind here
PLACE THIS CONSTRUCTIN CELLS THAT MAKE
THE PIT-1 PROTEIN
TO
TA
L L
IGH
T G
EN
ER
AT
ED
CELLEXRACTS
ADD ATP
MUTANTPROMOTER: TATGCCA
NORMALPROMOTER: TATGCAA
These LUCIFERASE-containing artificial geneswere placed inside cells that made the Pit-1transcription factor.
But if there were MUTATIONS in the promoter,very small amounts of luciferase were made.
The Pit-1 transcription factor was not able toproperly bind to the mutant promoter.
No promoter
in vectorATGCAA
TATGCAAin promoter
TATGCCAin promoter
Mutationelsewherein promoter
LU
CIF
ER
AS
EE
XP
ES
SIO
N
CONCLUSION:
This construct, containing the Luc gene, was placedinside cells that contained the Pit-1 transcription factor.
HOWEVER: a small single base-substitution in thepromoter stopped Pit-1 from binding, and Luc mRNAwas not transcribed from the construct. Correspondingly,the cells made much less luciferase.
THIS TECHNIQUE ALLOWED THE INVESTIGATORSTO FIND THE CRITICAL MUTATION THAT CAUSEDLOW LEVELS OF HGH TO BE RELEASED.
Most of what we know about transcription factor interactionswith promoters comes from studies like this with“artificial” chromosomes, with variations in the promoter.