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 DNAavailable forprotein bindingShown in red- groupsCan 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 MotifComparison 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 ofthe 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 proteinoperonsTranslation Repressor

Stringent response in E. coli – amino acid starvationuncharged tRNA binds to A siteRelA action ppGpp as starvation signal and regulate ~200 genes and rRNA

Salmonella typhimurium with flagella

Flagellin (MW 53 kD)are the targets of mammalianImmune system

Phase VariationSwitch between two distinct flagellin (FljB, FljC) once 1000generations

Regulation of flagellin genes in Salmonella : phase variationto 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

splice-site 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 transactivatorsDNA-binding domain and activation domain

DNA-binding transactivatorsDNA-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 eggAn 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 hasTwo 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