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Eukaryotic Regulation. Chapter 17 Sections:17.2, 17.3 - 17.7 &17.9. Eukaryotic Regulation Differs from Prokaryotic Regulation. Eukaryotes contain much greater amounts of genetic information Many chromosomes - PowerPoint PPT Presentation
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Chapter 17: Eukaryotic Gene Expression 1
Eukaryotic RegulationChapter 17
Sections:17.2, 17.3 - 17.7 &17.9
Chapter 17: Eukaryotic Gene Expression 2
Eukaryotic Regulation Differs from Prokaryotic Regulation
Eukaryotes contain much greater amounts of genetic information
Many chromosomes Genetic information is segregated from nucleus to
cytoplasm; Prokaryotes use cytoplasm only Posttranscriptional Regulation Eukaryotic mRNA has longer half-life Eukaryotic mRNA is more stable
Chapter 17: Eukaryotic Gene Expression 3
Types of Gene Regulation Control of Gene Expression
Chromosomal Organization Chromatin Remodeling
Transcription Promoters Enhancers (enhanceosome) Upstream Activating Sequences (UAS) Transcription Initiation Complex Activators
Chapter 17: Eukaryotic Gene Expression 4
Control of Gene Expression (continued)
mRNA Degradation Translational Control
RNA Silencing RNAi
mRNA Processing Alternative splicing
Chapter 17: Eukaryotic Gene Expression 5
Chapter 17: Eukaryotic Gene Expression 6
Transcription Control
Chapter 17: Eukaryotic Gene Expression 7
Transcriptional Control Why do you need a promoter?
Recognition site for binding of RNA polymerase Necessary for initiation of transcription Upstream from gene start site Several hundred nucleotides in length
Chapter 17: Eukaryotic Gene Expression 8
Transcriptional Control Actual Promoter : TATA BOX (-25 to –35) Sequences within the promoter region that
function as enhancers are:
1. CAAT or CCAAT (cat box)
-70 to –80
2. GGGCGG (GC box) -110
Chapter 17: Eukaryotic Gene Expression 9
Initiation Complex for Transcription
1. TFIID has 2 subunits : TBP and TAF
2. First, TBP subunit binds to TATA box
3. TAF promotes a conformational change in the DNA which allows other TF to bind (commitment stage)
4. Pol II leaves TATA box and transcribes (promoter clearance)
Chapter 17: Eukaryotic Gene Expression 10
Chapter 17: Eukaryotic Gene Expression 11
Chapter 17: Eukaryotic Gene Expression 12
Enhancers1. Necessary for full level of transcription
2. Responsible for tissue-specific gene expression
3. Able to bind transcription factors by associating with RNA polymerase forming DNA loops
Chapter 17: Eukaryotic Gene Expression 13
EnhancersDifferent from Promoters because:
1. No fixed position – upstream, downstream or within gene
2. Different orientation
3. Affect transcription of other genes if moved to another location
Chapter 17: Eukaryotic Gene Expression 14
Chapter 17: Eukaryotic Gene Expression 15
Positive Transcription Factors(True Activators)
A. Proteins with at least two functional domainsB. Functional Domains:
1. Bind to the enhancer (DNA binding domain)2. Protein-Protein interaction with RNA Pol or other transcription factors (trans-activating domain)
Chapter 17: Eukaryotic Gene Expression 16
Chapter 17: Eukaryotic Gene Expression 17
Positive Transcription Factors (True Activators)
DNA Binding Domains1. Helix-Turn-Helix (homeodomain) – 180 kb or 60 amino acids/ bind to major and minor grooves as well as backbone2. Zinc Fingers – Cys and His covalently bind zinc atom/bind major and minor goove Cys – N 2-4 - Cys – N 12-14 –His – N3 – His
Chapter 17: Eukaryotic Gene Expression 18
Helix-Turn-Helix
Chapter 17: Eukaryotic Gene Expression 19
Zinc Finger
Chapter 17: Eukaryotic Gene Expression 20
Zinc Finger
Chapter 17: Eukaryotic Gene Expression 21
Positive Transcription Factors (True Activators)
3. Leucine Zipper – 4 leucine residues spaced 7 amino acids apart and flanked by basic amino acids
- leucine regions form -helix
- leucine regions dimerize and and zip together
Chapter 17: Eukaryotic Gene Expression 22
Leucine Zipper
Chapter 17: Eukaryotic Gene Expression 23
Transcription Control
Chapter 17: Eukaryotic Gene Expression 24
Transcription Control: GAL genes Galactose-utilizing genes Part of metabolic pathway to metabolize galactose in
yeast Follow the activation of genes GAL 1, 7, 10 that are
located near one another on the DNA Genes are made in response to the presence of
galactose Gal4p and Gal80p are regulatory proteins in the
process and UAS-G is the DNA sequence
Chapter 17: Eukaryotic Gene Expression 25
Transcription Control: GAL genes
Chapter 17: Eukaryotic Gene Expression 26
Transcription Control: GAL genes In the absence of galactose, GAL 80p is bound to
GAL 4p and GAL 4p is bound to the regulatory DNA sequence (UAS-G) Under these conditions, transcription of GAL 1, 7, 10 is
inhibited In the presence of galactose, a metabolite of
galactose binds to GAL 80p GAL 4p is then phosphorylated initiating a change in
conformation GAL 4p is now capable of activating transcription
Chapter 17: Eukaryotic Gene Expression 27
Control of GAL Genes
Chapter 17: Eukaryotic Gene Expression 28
Transcription Control: GAL genes
Fig. 17.5
Chapter 17: Eukaryotic Gene Expression 29
GAL Genes
Chapter 17: Eukaryotic Gene Expression 30
Transcription Control: Steroid Hormone
Not many changes in the external environment of cell in an animal
Hormones are secreted by cells in the animal and can signal changes from the environment
Peptide hormones bind to extra cellular receptors and steroid hormones bind to intracellular receptors
Chapter 17: Eukaryotic Gene Expression 31
Transcription Control: Steroid Hormone
Chapter 17: Eukaryotic Gene Expression 32
Transcription Control: Steroid Hormone
Steroid hormones often bind to cytoplasmic receptor and translocated to the nucleus where the complex acts
In the nucleus the complex binds to the DNA at a specific sequence
Hormones are potent regulators of gene expression, but only affect cells that produce the receptor that the particular hormone binds
Chapter 17: Eukaryotic Gene Expression 33
Transcription Control: Steroid Hormone
Chapter 17: Eukaryotic Gene Expression 34
Transcription Control: Steroid Hormone
Chapter 17: Eukaryotic Gene Expression 35
Transcription Control: Steroid Hormone Steroid hormone control of gene expression
Important in development and physiological regulation
Because receptor is needed, have tissue or cell type specific effects
Specific for certain hormone receptor Usually found in a small number of cells Can affect tc, mRNA stability, mRNA processing
Chapter 17: Eukaryotic Gene Expression 36
Transcription Control: Steroid Hormone Steroid hormone control of gene expression
No hormone then the receptor is inactive and bound to a chaperone protein
Steroid hormone enters cell and binds to its specific receptor
Chaperone is displaced Hormone binds receptor = activation Complex is transported and acts in the nucleus
Chapter 17: Eukaryotic Gene Expression 37
Transcription Control: Steroid Hormone Steroid hormone control of gene expression
Hormone-receptor complex binds to specific DNA binding element Transcription activation or repression depending on
the complex Complex binds to the steroid hormone response
element (HRE) in the DNA HRE’s are in the enhancer region and in multiple
copies
Chapter 17: Eukaryotic Gene Expression 38
Transcription Control Transcription of a gene is also affected by the
proteins bound to the DNA (histones) DNA is less compacted in regions where DNA is
transcribed Nucleosomes are not removed Generally physically inhibit gene transcription Transcription can occur in the presence of
nucleosomes when they are chemically modified DNA Methylation – CpG islands/X chromosome
Chapter 17: Eukaryotic Gene Expression 39
Control of mRNA mRNA processing—regulation of production
of mature mRNA Alternative poly-A sites Alternative/differential splicing CALC gene employs both in different cell types
Chapter 17: Eukaryotic Gene Expression 40
Control of mRNA
Fig. 17.7
Chapter 17: Eukaryotic Gene Expression 41
Control of mRNA Evaluate gene expression of the human
calcitonin gene (CALC) in thyroid cells and neurons.
Thyroid cells Poly(A) signal after exon 4 is used Removed introns 1-4 and join exons 1-4 to make
calcitonin mRNA is translated.
Chapter 17: Eukaryotic Gene Expression 42
Control of mRNA Evaluate gene expression of the human
calcitonin gene (CALC) in thyroid cells and neurons.
Neurons Poly(A) signal after exon 5 is used Remove all introns and exon 4 is removed as
well; join exons 1, 2, 3, 5 to make CGRP mRNA mRNA is translated.
Chapter 17: Eukaryotic Gene Expression 43
Posttranslational modification Evaluate gene expression of the human
calcitonin gene (CALC) in thyroid cells and neurons. In both cell types the mRNA is translated into a
protein that needs processing—pre-hormone or pre-protein
This allows the protein to be synthesized and be present in the cell, but NOT be active.
Chapter 17: Eukaryotic Gene Expression 44
Posttranslational modification When the proteins are needed, a protease cleaves
the pre-portion of the protein and the remainder of the polypeptide becomes active Calcitonin is produced in thyroid cells—hormone that
helps the kidney to retain calcium; Exon 4 encodes the active protein
cGRP is produced in neurons—found in hypothalamus and has neuromodulary/growth promoting properties; Exon 5 encodes the active protein
Chapter 17: Eukaryotic Gene Expression 45
Control of Translation Shortened poly(A) tails prevent translation
Poly(A) tails are needed for translation initiation mRNAs that are ‘stored’ and prevented from
being translated have short Poly(A) tails (15-90 A’s long)
Chapter 17: Eukaryotic Gene Expression 46
Control of Translation Shortened poly(A) tails prevent translation
Tails may be trimmed (deadenylation enzymes) or they may be short at synthesis.
Deadenylation enzymes recognize AU rich element (ARE) in the 3’ UTR of the mRNA and remove A’s from the tail
Other enzymes may recognize ARE in the 3’ UTR and lengthen the poly(A) tail when it is time to translate the mRNA
Chapter 17: Eukaryotic Gene Expression 47
Control of mRNA mRNA stability—how long the mRNA is
found in the cell (RNA turnover) The longer the mRNA is found in the cell, the
more copies of protein are made. Stability of mRNA varies greatly from gene to
gene Important way to control gene expression
Chapter 17: Eukaryotic Gene Expression 48
Control of mRNA mRNA stability—how long the mRNA is
found in the cell (RNA turnover) Stability can be controlled by molecules present
in the cell Signals found in the 5’ or 3’ UTR Control when the mRNA is degraded
Chapter 17: Eukaryotic Gene Expression 49
Control of mRNA mRNA stability—how long the mRNA is
found in the cell (RNA turnover) 2 major pathways
Deadenylation –dependent decay pathway Deadenylation-independent decay pathway
Chapter 17: Eukaryotic Gene Expression 50
Control by Protein Degradation Posttranslational control Controls how long the protein is present and active
in the cell Controlled by attachment of the protein ubiquitin to
the protein being targeted for degradation Signals for the protein to be degraded by the
proteasome N-terminus of the protein will determine its stability
by determining the rate that ubiquitin can bind to the protein