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

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

DNA Level (transcription control)

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-transcriptionComplex

RNA Synthesis occurs on chromatin, not DNA. Chromatin is a protein DNA complex

TAF

Pol II

IIHIIEIIF

IIBTBPIIA MRP

Act

TATA

Enhancers

NucleosomeActivation domain (recruits other proteins)

DNA binding domain

mRNA Metal-responsive protein transcription

factor

Zn ZnZn

E2F

Rb

Cyclins Cell proliferation

CDK

Cyclin genesPromoter

Rb = retinoblastoma protein (tumor suppressor)

P

Cancer of the retina

E2F = a transcription factor that stimulates transcription of cyclin genes

ADP

ATP

Cancer of the Retina

Activates

Stimulates CDK

Inactive

Phosphate controls cell division

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 zinc-thionein 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

mRNA for metallothionein

Metallothionein (binds excess cytosolic Zinc)

DNAMRE

Zn

Transcription factor for Zinc

Zn

Zn

Inhibited by Actinomycin D

X

Zn induces Metallothionein

35S-Cystine

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 Cd-thionein. Cycloheximide and actinomycin D prevented the synthesis of thionein. The concentration of Cd affected the amount of Cd-thionein synthesized.

Cd

mRNA for metallothionein

Metallothionein (binds excess cytosolic Zinc)

DNAMRE

Cd

Transcription factor for Zinc

Cd

Cd

Inhibited by Actinomycin D

X

Cd induces Metallothionein

35S-Cystine

Cycloheximide

X

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.

Transcription Start Site-42

-60

DNA

Upstream Downstream

Promoter Region of the metallothein

gene

MRE 12 bp

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.

Transcription Start Site

-200

DNA

Upstream Downstream

Promoter Region of the metallothein

gene

MRE-1MRE-2MRE-3MRE-4

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

Conclusions

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