regulation of gene expression
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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
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