Transcript Lecture 15 Genetic Regulation

A Role for Minerals in Genetic
Expression
Summary:
Metal ions are not passive components of biological systems
as once thought. Recent studies have shown that Fe, Cu, or
Zn are capable of regulating the expression of genes that code
for that metal’s storage or transport proteins
How should gene regulation be viewed
DNA Level (transcription control)
Transcription factors bind to the promoter region
and turn on the downstream gene
Base sequences (MRE’s) in promoter form the
binding site
The presence of the metal is needed for binding to occur
RNA Level (translation control)
5’-sequences determine whether the mRNA will be read
3’-sequences stabilize mRNA to turnover
Both engage proteins that are activated (deactivated)
by the metal
Pre-transcription
Complex
RNA Synthesis occurs on chromatin,
not DNA. Chromatin is a protein DNA complex
DNA binding domain
Enhancers
Act
Activation domain (recruits other proteins)
Nucleosome
TAF
IIA TBP IIB
IIF
IIE
IIH
MRP
Pol II
TATA
mRNA
Zn
Zn
Zn
Metal-responsive
protein transcription
factor
Cancer of the Retina
Promoter
Cyclin genes
Activates
E2F
P
Inactive
Rb
ADP
CDK
ATP
Cyclins
Cell proliferation
Stimulates
CDK
Cancer of the retina
Phosphate controls cell
division
E2F = a transcription factor that stimulates transcription of cyclin genes
Rb = retinoblastoma protein (tumor suppressor)
Early Observations that
Established Metal Ion
Regulation of Genes
Biochem J. 1977 Apr 15;164(1):223-8.
Control of zinc-thionein synthesis in rat
liver.
Squibb KS, Cousins RJ, Feldman SL.
The rate of [35S]cystine incorporation into hepatic
zinc-thionein (a metallothionein) was stimulated after
parenteral administration of 2 mg of Zn2+.
Stimulation was inhibited by actinomycin D or
cordycepin, supporting the concept that hepatic zincthionein synthesis responds quickly to changes in
Zn2+ status that allow the Zn2+ to bind to the
nascent thionein chains. The control of zinc-thionein
synthesis by Zn2+ appears to involve changes in
RNA
Zn induces Metallothionein
Inhibited by Actinomycin D
MRE
X
DNA
mRNA for metallothionein
Transcription factor
for Zinc
Zn
Zn
Zn
Zn
35S-Cystine
Metallothionein
(binds excess
cytosolic Zinc)
Biochem J. 1978 Feb 15;170(2):219-225.
Induction of cadmium-thionein in isolated rat liver
cells.
Hidalgo HA, Koppa V, Bryan SE.
The uptake of Cd by isolated liver cells was
linearly related to the Cd concentration to which the
cells were exposed. Cd-treated cells synthesized
proteins de novo with the characteristics of Cdthionein. Cycloheximide and actinomycin D prevented
the synthesis of thionein. The concentration of Cd
affected the amount of Cd-thionein synthesized.
Cd induces Metallothionein
Inhibited by Actinomycin D
MRE
DNA
X
mRNA for metallothionein
Transcription factor
for Zinc
Cd
Cd
Cd
Cd
X
35S-Cystine
Metallothionein
(binds excess
cytosolic Zinc)
Cycloheximide
Biochem J. 1978 Dec 1;175(3):833-40.
Cell-free synthesis of metallothionein directed by rat
liver polyadenylated messenger ribonucleic acid.
Shapiro SG, Squibb KS, Markowitz LA, Cousins RJ.
Polyribosomal metallothionein mRNA was
increased at least 3-fold after of Zn2+ administration.
Actinomycin D blocked the increase strongly
suggesting that Zn2+ caused an elevation in
metallothionein mRNA.
-60
DNA
-42
Transcription
Start Site
MRE
12 bp
Upstream
Promoter Region of
the metallothein
gene
Downstream
bp = base pairs
Nucleic Acids Res. 1990 August 25; 18(16): 4683–
4690.
Zinc dependent binding of a liver nuclear factor
to metal response element MRE-a of the mouse
metallothionein-I gene and variant sequences.
P F Searle
Metallothionein gene transcription is
inducible by zinc and other heavy metals,
and several metal response elements (MREs)
have been mapped within about 200 bp
upstream of the site of transcription initiation
in several metallothionein genes.
-200
DNA
MRE-4 MRE-3
Transcription
Start Site
MRE-2
Upstream
Promoter Region of
the metallothein
gene
MRE-1
Downstream
Conclusions
Sequences in the DNA promoter for
metallothionein are specific for proteins that
serve as metal-activated transcription factors
Zn-specific transcription factors are
activated by Zn2+, resulting in a
prioritized synthesis of the mRNA coding
for metallothionein protein
What about Copper?
Proc Natl Acad Sci U S A. 1991 October 1; 88(19): 8558–
8562.
ACE1, a copper-dependent transcription factor, activates
expression of the yeast copper, zinc superoxide
dismutase gene.
E B Gralla, D J Thiele, P Silar, and J S Valentine
In the yeast Saccharomyces cerevisiae, copper
levels exert some control over the level of SOD1
expression. We show that the ACE1 transcriptional
activator protein, which is responsible for the
induction of yeast metallothionein (CUP1) in response
to copper, also controls the SOD1 response to copper.
Proc Natl Acad Sci U S A. 1976 Feb;73(2):371-4.
Copper-induced activation of aortic lysyl oxidase in
vivo.
Harris ED.
Day-old chicks lacking Cu had severely
depressed lysyl oxidase, a Cu metalloenzyme.
CuSO4 restored lysyl oxidase activity in aortic
tissue. 2 hours after receiving CuSO4 (1 mg/kg) lysyl
oxidase activity rose to a level 5-20 times higher
than basal (saline-injected) activity. 20 hours
doubled the activity. Cycloheximide suppressed
activation by two-thirds. Actinomycin D caused only
a 10-15% inhibition. The data suggest that Cu
regulates lysyl oxidase activity and may determined
its steady-state levels.
Metals Ions as Suppressors of Genetic
Expression
A regulatory as opposed to toxic response
Proc Natl Acad Sci U S A. 2006 Sep 12;103(37):13676-81. Epub
2006 Aug 31.
NikR-operator complex structure and the mechanism of
repressor activation by metal ions.
Schreiter ER, Wang SC, Zamble DB, Drennan CL.
Regulation of nickel concentrations in Escherichia coli is
mediated by the NikR repressor via nickel-induced
transcriptional repression of the nickel ABC-type transporter,
NikABCDE.
Biochemistry. 2006 Mar 21;45(11):3493-505.
Structural basis for the metal-selective activation of the
manganese transport regulator of Bacillus subtilis.
Kliegman JI, Griner SL, Helmann JD, Brennan RG, Glasfeld A.
The manganese transport regulator (MntR) of Bacillus
subtilis is activated by Mn(2+) to repress transcription of genes
encoding transporters involved in the uptake of manganese. MntR
is also strongly activated by cadmium, both in vivo and in vitro, but
it is poorly activated by other metal cations, including calcium and
zinc.
Many transcription factors are Zn-Finger proteins
Metals at the Post-transcriptional Level
Regulation at the level of RNA