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3/18/2014
1
Mechanisms of Eukaryotic Gene Regulation
2 Terms before we get started: Types of Chromatin
• Heterochromatin:
– Highly condensed
– Not expressed = Off!
• Euchromatin:
– Less condensed
– Available for transcription = On!
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Fig. 16-21a
DNA double helix (2 nm in diameter)
Nucleosome (10 nm in diameter)
Histones Histone tail
H1
DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)
Fig. 16-21b
30-nm fiber
Chromatid (700 nm)
Loops Scaffold
300-nm fiber
Replicated chromosome (1,400 nm)
30-nm fiber Looped domains (300-nm fiber)
Metaphase chromosome
Animation embedded
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Fig. 18-6
DNA
Signal
Gene
NUCLEUS
Chromatin modification
Chromatin
Gene available
for transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
mRNA in cytoplasm
Translation
CYTOPLASM
Degradation
of mRNA
Protein processing
Polypeptide
Active protein
Cellular function
Transport to cellular
destination
Degradation
of protein
Transcription
Prokaryotic gene regulation
is at the level of transcription,
for the most part =
The Operons!
Eukaryotic gene regulation
occurs at multiple levels –
see this figure. ALL the
boxes represent possible
control points.
Fig. 18-7
Histone tails
DNA
double helix
(a) Histone tails protrude outward from a nucleosome
Acetylated histones
Amino acids available for chemical modification
(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription
Unacetylated histones
Histone Acetylation
(Chromatin modification Mechanism)
Acetyl groups = turn on!
De-acetylate = turn off!
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DNA Methylation
• DNA methylation, the addition of methyl groups to certain bases in DNA (usually cytosine), is associated with reduced transcription in some species
• DNA methylation can cause long-term inactivation of genes in cellular differentiation
(Chromatin modification mechanism)
Methyl groups = turn off!
De-methylate = turn on!
Epigenetic Inheritance
• Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells
• The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance
• (…watching a video on this later this week…)
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Fig. 18-6
DNA
Signal
Gene
NUCLEUS
Chromatin modification
Chromatin
Gene available
for transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
mRNA in cytoplasm
Translation
CYTOPLASM
Degradation
of mRNA
Protein processing
Polypeptide
Active protein
Cellular function
Transport to cellular
destination
Degradation
of protein
Transcription
The Roles of Transcription Factors
• To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors
• General transcription factors are essential for the transcription of all protein-coding genes
• In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors
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• Proximal control elements are located close to the promoter
• Distal control elements, groups of which are called enhancers, may be far away from a gene or even located in an intron
Enhancers and Specific Transcription Factors
• An activator is a protein that binds to an enhancer and stimulates transcription of a gene
• Bound activators cause mediator proteins to interact with proteins at the promoter
Fig. 18-9-3
Enhancer TATA box
Promoter Activators
DNA Gene
Distal control element
Group of mediator proteins
DNA-bending protein
General transcription factors
RNA polymerase II
RNA polymerase II
Transcription initiation complex RNA synthesis
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Fig. 18-10
Control elements
Enhancer
Available activators
Albumin gene
(b) Lens cell
Crystallin gene expressed
Available activators
LENS CELL NUCLEUS
LIVER CELL NUCLEUS
Crystallin gene
Promoter
(a) Liver cell
Crystallin gene not expressed
Albumin gene expressed
Albumin gene not expressed
Fig. 18-6
DNA
Signal
Gene
NUCLEUS
Chromatin modification
Chromatin
Gene available
for transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
mRNA in cytoplasm
Translation
CYTOPLASM
Degradation
of mRNA
Protein processing
Polypeptide
Active protein
Cellular function
Transport to cellular
destination
Degradation
of protein
Transcription
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RNA Processing
• In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns
Fig. 18-11
or
RNA splicing
mRNA
Primary RNA transcript
Gene – introns and exons
Exons
DNA
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Fig. 18-6
DNA
Signal
Gene
NUCLEUS
Chromatin modification
Chromatin
Gene available
for transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
mRNA in cytoplasm
Translation
CYTOPLASM
Degradation
of mRNA
Protein processing
Polypeptide
Active protein
Cellular function
Transport to cellular
destination
Degradation
of protein
Transcription
mRNA Degradation
• The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis
• Eukaryotic mRNA is more long lived than prokaryotic mRNA
• The mRNA life span is determined in part by sequences in the leader and trailer regions – UTR’s
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Fig. 18-6
DNA
Signal
Gene
NUCLEUS
Chromatin modification
Chromatin
Gene available
for transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
mRNA in cytoplasm
Translation
CYTOPLASM
Degradation
of mRNA
Protein processing
Polypeptide
Active protein
Cellular function
Transport to cellular
destination
Degradation
of protein
Transcription
Protein Processing and Degradation
• After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control
• Proteasomes are giant protein complexes that bind protein molecules and degrade them
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Fig. 18-12
Proteasome and ubiquitin to be recycled Proteasome
Protein fragments (peptides) Protein entering a
proteasome
Ubiquitinated protein
Protein to be degraded
Ubiquitin
Fig. 18-6
DNA
Signal
Gene
NUCLEUS
Chromatin modification
Chromatin
Gene available
for transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
mRNA in cytoplasm
Translation
CYTOPLASM
Degradation
of mRNA
Protein processing
Polypeptide
Active protein
Cellular function
Transport to cellular
destination
Degradation
of protein
Transcription
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Concept 18.3: Noncoding RNAs play multiple roles in controlling gene expression
• Only a small fraction of DNA codes for proteins, rRNA, and tRNA
• A significant amount of the genome is transcribed into noncoding RNAs
• Noncoding RNAs regulate gene expression at two points: degrade mRNA or block its translation
• MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to mRNA
• The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi)
• RNAi is caused by small interfering RNAs (siRNAs)
• siRNAs and miRNAs are similar but form from different RNA precursors
• Prokaryotic and eukaryotic cells use small RNA’s to regulate gene expression
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Fig. 18-13
miRNA- protein complex (a) Primary miRNA transcript
Translation blocked
Hydrogen bond
(b) Generation and function of miRNAs
Hairpin miRNA
miRNA
Dicer
3
mRNA degraded
5
2 ways that miRNA’s
interfere with gene
expression:
Summary of Several Eukaryotic Gene Regulation Mechanisms
• Genes in highly compacted
chromatin are generally not
transcribed.
Chromatin modification
• DNA methylation generally reduces transcription.
• Histone acetylation seems to
loosen chromatin structure,
enhancing transcription.
Chromatin modification
Transcription
RNA processing
Translation mRNA degradation
Protein processing and degradation
mRNA degradation
• Each mRNA has a characteristic life span, determined in part by sequences in the 5’ and 3’ UTRs.
• Protein processing and degradation by proteasomes are subject to regulation.
Protein processing and degradation
• Initiation of translation can be controlled via regulation of initiation factors.
Translation
or mRNA
Primary RNA transcript
• Alternative RNA splicing:
RNA processing
• Coordinate regulation:
Enhancer for
liver-specific genes
Enhancer for
lens-specific genes
Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription.
Transcription
• Regulation of transcription initiation: DNA control elements bind specific transcription factors.
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Summary Roles of Small RNA’s in Gene Regulation of Eukaryotic Cells
Chromatin modification
RNA processing
Translation mRNA degradation
Protein processing
and degradation
mRNA degradation
• miRNA or siRNA can target specific mRNAs
for destruction.
• miRNA or siRNA can block the translation
of specific mRNAs.
Transcription
• Small RNAs can promote the formation of
heterochromatin in certain regions, blocking
transcription.
Chromatin modification
Translation
Concept 18.5: Cancer results from genetic changes that affect cell cycle
control
• The gene regulation systems that go wrong during cancer are the very same systems involved in embryonic development
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Oncogenes and Proto-Oncogenes
• Oncogenes are cancer-causing genes
– Analogy: Foot on the gas pedal!
• Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division
• Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle
– Analogy: Putting the pedal to the metal!
Fig. 18-20
Normal growth- stimulating protein in excess
DNA
Proto-oncogene
Normal growth-stimulating protein in excess
Normal growth- stimulating protein in excess
Hyperactive or degradation- resistant protein
Point mutation:
Oncogene Oncogene
within a control element within the gene
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Tumor-Suppressor Genes • Tumor-suppressor genes help prevent uncontrolled
cell growth
– Analogy: Foot on the brake pedal!
• Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset
– Analogy: Taking the foot of the brake pedal!
• Tumor-suppressor proteins
– Repair damaged DNA
– Control cell adhesion
– Inhibit the cell cycle in the cell-signaling pathway
• Suppression of the cell cycle can be important in the case of damage to a cell’s DNA
• The p53 gene prevents a cell from passing on mutations due to DNA damage
• Mutations in the p53 gene prevent suppression of the cell cycle
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Interference with Normal Cell-Signaling Pathways …add to lecture…
• Mutations in the ras proto-oncogene and p53 tumor-suppressor gene are common in human cancers
• Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division
Fig. 18-21a
Receptor
Growth factor
G protein GTP
Ras
GTP
Ras
Protein kinases (phosphorylation cascade)
Transcription factor (activator)
DNA
Hyperactive Ras protein (product of oncogene) issues signals on its own
MUTATION
NUCLEUS
Gene expression
Protein that stimulates the cell cycle
(a) Cell cycle–stimulating pathway
1 1
3
4
5
2
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Fig. 18-21b
MUTATION
Protein kinases
DNA
DNA damage in genome
Defective or
missing
transcription
factor, such
as p53, cannot
activate
transcription
Protein that
inhibits
the cell cycle
Active form of p53
UV light
(b) Cell cycle–inhibiting pathway
2
3
1
Fig. 18-21c
(c) Effects of mutations
EFFECTS OF MUTATIONS
Cell cycle not
inhibited
Protein absent
Increased cell
division
Protein
overexpressed
Cell cycle
overstimulated
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The Multistep Model of Cancer Development
• Multiple mutations are generally needed for full-fledged cancer; thus the incidence increases with age
• At the DNA level, a cancerous cell is usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes
Fig. 18-22
EFFECTS OF MUTATIONS
Malignant tumor
(carcinoma)
Colon
Colon wall
Loss of tumor-
suppressor gene Activation of
oncogene
Loss of
tumor-suppressor
gene
Loss of
tumor-suppressor
gene p53
Additional
mutations
Larger benign
growth (adenoma)
Small benign
growth (polyp)
Normal colon
epithelial cells
5
4 2
3
1
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Summary of Several Eukaryotic Gene Regulation Mechanisms
• Genes in highly compacted
chromatin are generally not
transcribed.
Chromatin modification
• DNA methylation generally reduces transcription.
• Histone acetylation seems to
loosen chromatin structure,
enhancing transcription.
Chromatin modification
Transcription
RNA processing
Translation mRNA degradation
Protein processing and degradation
mRNA degradation
• Each mRNA has a characteristic life span, determined in part by sequences in the 5’ and 3’ UTRs.
• Protein processing and degradation by proteasomes are subject to regulation.
Protein processing and degradation
• Initiation of translation can be controlled via regulation of initiation factors.
Translation
or mRNA
Primary RNA transcript
• Alternative RNA splicing:
RNA processing
• Coordinate regulation:
Enhancer for
liver-specific genes
Enhancer for
lens-specific genes
Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription.
Transcription
• Regulation of transcription initiation: DNA control elements bind specific transcription factors.
Summary Roles of Small RNA’s in Gene Regulation of Eukaryotic Cells
Chromatin modification
RNA processing
Translation mRNA degradation
Protein processing
and degradation
mRNA degradation
• miRNA or siRNA can target specific mRNAs
for destruction.
• miRNA or siRNA can block the translation
of specific mRNAs.
Transcription
• Small RNAs can promote the formation of
heterochromatin in certain regions, blocking
transcription.
Chromatin modification
Translation
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