Regulation of Gene Expression
David Shiuan
Department of Life Science
Institute of Biotechnology
Interdisciplinary Program of Bioinformatics
National Dong Hwa University
The fundamental problem of chemical
physiology and of embryology --- is to
understand why tissue cells do not all
express, all the time, all the potentialities
inherent in their genome.
Francois Jacob and Jacques Monod
J. Mol. Biol. 1961
• 1. Principle of gene regulation
• 2. Regulation of gene expression in
prokaryotes
• 3. Regulation of gene expression in
eukaryotes
Seven processes affect the steady-state
concentration of a protein
Potential Points of Regulation
• Synthesis of primary RNA transcript
(transcription)
• Posttranscriptional modification of mRNA
• mRNA degradation
• Protein synthesis (translation)
• Posttranslational modification of proteins
• Protein targeting and transport
• Protein degradation
1. Principle of gene regulation
Molecular circuits ------------------------------House keeping genes; constitutive gene expression
Inducible; induction; repressible; repression
RNA polymerase binds to DNA at promoters
Consensus sequence for promoters that regulate
expression of the E. coli heat shock genes
Many prokaryotic genes are clustered and regulated in operons
Lactose
metabolism
in E. coli
They published a paper - Coordinated regulation
of lac operon, Proc. French Acad. Sci. (1960)
The lac operon
Lac repressor binds to operator O2 and O3
Lac repressor binds to operator (PDB-1BLG)
Lac repressor binds to operator - Conformational
change in the repressor caused by DNA binding
Lac inducer IPTG, structurally similar to lactose
Groups in DNA
available for
protein binding
Shown in red- groups
Can recognize proteins
Protein-DNA interactions
Relationship between the lac operator sequence O1
and the lac promoter
DNA Binding Domain of Lac Repressor
- Helix-turn-helix
Surface rendering of the DNA-binding domain
gray - lac repressor; blue - DNA
The DNA-binding domain, but separated
The zinc-finger – each Zn2+ coordinates
with 2 His and 2 Cys residues
Homeodomain - approx. 60 aa
Homeotic genes (genes that regulate the development of body patterns)
DNA-Binding Domain - helix-turn-helix
Studying DNA-Protein Interactions
• EMSA (electrophoretic mobility shift assay); or
gel retardation assay
• DNaseI footprinting experiment
• DNA affinity chromatography
• SPR (Surface Plasmon Resonance)/BIACORE
• CD/ORD; Spefctrofluorometry; NMR
EMSA- M. hyopneumoniae
HrcA-CIRCE Interaction
1
2
3
4
5
DNaseI Footprinting – JBBM 30 (1995) 85-89
DNA Affinity Chromatography
Surface Plasmon Resonance (SPR)
• SPR - Surface plasmon resonance is a phenomenon
which occurs when light is reflected off thin metal films.
A fraction of the light energy incident at a sharply defined
angle can interact with the delocalized electrons in the
metal film (plasmon) thus reducing the reflected light
intensity
DNA-Binding Motif
Comparison of aa sequences of several leucine zipper proteins
Leucine zipper from yeast activator protein (1YSA)
Helix-loop-helix – the human transcription
factor Max, bound to DNA target 1HLO
2. Regulation of Gene Expression in Prokaryotes
Catabolic Repression - restricts expression of
the genes required for catabolism of lactose, arabinose
and other sugar in the presence of glucose
CRP (cAMP Receptor Protein) homodimer
- bound with cAMP
The trp Operon
The trp Repressor
Transcriptional attenuation in the trp operon
SOS response in E. coli
- RecA/ssDNA cleaves repressor LexA
Translational feedback
in some ribosomal protein
operons
Translation Repressor
Stringent response in E. coli – amino acid starvation
uncharged tRNA binds to A siteRelA action  ppGpp as
starvation signal and regulate ~200 genes and rRNA
Salmonella typhimurium with flagella
Flagellin (MW 53 kD)
are the targets of mammalian
Immune system
Phase Variation
Switch between two distinct
flagellin (FljB, FljC) once 1000
generations
Regulation of flagellin genes in Salmonella : phase variation
to evade the host immune response
3. Regulation of Gene Expression in Eukaryotes
Eukaryote Gene Regulation
- Four Different Features
1. Eukaryotic promoter is restricted by the
structure of chromatid
2. Positive regulation
3. More multimeric regulatory proteins
4. Transcription is separated from
translation in both space and time
Transcriptionally Active Chromatin is
Structurally Distinct from Inactive Chromatin
• Heterochromatin - ~10% in eukaryotic cells, more
condensed, transcriptionally inactive, generally associated
with chromosome structure such as centormeres
• Euchromatin - the remaining, less condensed chromatin
• Hypersensitive Sites - in actively transcribed regions;
many bind to regulatory proteins
• Histones – different modifications in different regions
Histones – different modifications in different regions
Nucleosome core proteins
Modification – methylation,
acetylation, attachment of
ubiquitin
Histones act as a general repressor of transcription,
because they interfere with protein binding to DNA
1. Histones form nucleosomes on TATA boxes, blocking
transcription. Promoter-binding proteins cannot disrupt the
nucleosomes. Enhancer-binding proteins bind to enhancers,
displacing any histones, and then cause the histones at the
TATA box to free the DNA.
2. Histone Acetylation with increased transcription.
Histone are acetylated on lysines in regions on the outside of
the nucleosome.
Acetylation destabilizes higher-order chromatin structure.
DNA becomes more accessible to transcription factors, and
overcoming histone repression of transcription.
DNA Methylation
1.
DNA methylation and transcription are correlated, with
lower levels of methylated DNA in transcriptionally
active genes.
2. Other recent observations also indicate a role for
methylation in gene expression:
(a) A methylase is essential for development in mice.
(b) Methylation is involved in fragile X syndrome, where
expansion of a triplet repeat and abnormal methylation
in the FMR-1 gene silence its expression.
Chromatin Remodeling – detailed mechanisms for
transcription-associated structural changes in chromatin
Acetylation in histone H3 globular domain
regulate gene expression in Yeast Cell 121 (2005) 375
• Lys 56 in histone H3 : in the
globular domain and extends
toward the DNA major
groove/nucleosome
• K56 acetylation : enriched at
certain active genes, such as
histones
Acetylation in histone H3 globular domain regulates
gene expression in yeast
Cell 121(2005) 375
• SPT10, a putative acetyltransferase: required
for cell cycle-specific K56 acetylation at histone
genes
• Histone H3 K56 acetylation at the entry-exit
gate enables recruitment of the SWI/SNF
nucleosome remodeling complex and so
regulates gene activity
The RNA degradosome
TIBS 31 (2006) 359-365
• Most mRNA molecules are destroyed shortly after
synthesized
• In E.coli, a multi-enzyme complex RNA degradosome
- can drive the energy-dependent turnover of mRNA
and trim RNA species into their active forms
• Degradosome comprises :
1. endoribonuclease RNase E : initiates the mRNA turnover
2. ATP-dependent RNA helicase RhlB : unwinds and translocates RNA
3. glycolytic enzyme : Enolase
4. phosphorolytic exoribonuclease : PNPase
(a) Structure of RNaseE/RNaseG
(b) The protein-RNA recognition domain
The structural information for components of the E. coli
RNA degradosome and a model of degradosome assembly
Alternative pre-mRNA splicing
TIBS 25 (2000) 381-388
Different modes of alternative splicing and its consequences
Splice-site
elements
and splicing
complex
assembly
Packing and Remodelling RNA
• Primary transcripts associate with a family of
polypeptides known as hnRNP proteins
• They contain RNA-binding motifs, and Gly-rich
domains for protein–protein interactions and RNA
transport
• hnRNP packaging can also bring together distant
regions of the pre-mRNA and therefore assist splicesite pairing
Post-transcriptional control of gene expression:
a genome-wide perspective
TIBS 30 (2005) 506-514
Many eukaryotic promoters
are positively regulated
• RNA polymerases have little or no intrinsic affinity for
promoters
• Transcription initiation depends on activator proteins
• Enhancer; Upstream Activator Sequence (UAS, Yeast)
• Basal transcription factors – RNA pol II
DNA-binding transactivator – enhancer
Coactivator – interconnections
Repressors -
Eukaryotic promoters and regulatory proteins
Eukaryotic transcriptional repressor
Galactose-utilization genes of yeast
GAL1 galactokinase. GAL7 galactose transferase.
GAL10 galactose epimerase
Regulation of galactose metabolism in yeast
Activation of Yeast Gal Genes
Protein complexes involved in transcription activation
of a group of related eukaryotic genes (yeast Gal system)
DNA-binding transactivators
DNA-binding domain and activation domain
DNA-binding transactivators
DNA-binding domain of Sp1 and the activator
domain of CTF1 activates transcription of a GC box
chimeric protein
Eukaryotic gene expression can be regulated by
intracellular and extracellular signals
• Steroid hormone – extracellular signal
bind to intracellular receptor hormone-receptor
complex binds to HRE (hormone-response elements)
• Regulation through phosphorylation of
transcription factors – intracellular signal
Typical steroid hormone receptors
Translational Regulation
1.
Phosphorylation of initiation factor  less
active
2.
Protein repressor bind to 3’UTR of mRNA to
prevent translation initiation
3.
Binding proteins disrupt the interaction of
elF4E and elF4G to prevent the formation of
eukaryotic initiation complex
Phosphorylation of
initiation factors
-Regulation of Gene
Expression by Insulin
Translational regulation of eukaryotic mRNA
(1) elF interactions
(2) 3’UTR binding
Post-transcriptional gene silencing
by RNA interference
(endonuclease)
Development is controlled by cascades of
regulatory proteins - life cycle of fruit fly
幼蟲
蛹
Development is controlled by
Cascades of Regulatory Proteins
• Polarity (anterior/posterior; dorsal/ventral)
• Metamerism (serially repeating segments)
• Pattern regulating genes – morphogens
1. maternal genes (expressed in unfertilized eggs)
2. segmentation genes (gap; pair-rule; segment
polarity)
3. homeotic genes (expressed later to organs)
Early development in Drosophila
Distribution of a maternal gene product in a Drosophila egg
An immunologically stained egg, showing the distribution of
bicoid (bcd) gene product
If bcd gene is not expressed by the mother (bcd- mutant) thus
No bcd mRNA is deposited in the egg, the resulting embryo has
Two posteriors (and soon die)
Regulatory circuits of the anterior-posterior axis in a Drosophila egg
Distribution of the fushi tarazu (ftz) gene product
in early Drosophila embryos
Homeotic Genes
• Homeotic genes - genes that regulate the development
of body patterns
• Homeodomain - approx. 60 aa; helix-turn-helix;
• Homeobox - DNA part
• Ultrabithrax (ubx) gene: 76 kb (73 kb intron)
Ubx protein is transcriptional activator
Effects of mutation in homeotic genes in Drosophila