Transcript CHAPTER 16 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

CHAPTER 16
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Control of Gene Expression
• Controlling gene expression is often
accomplished by controlling transcription
initiation
• Regulatory proteins bind to DNA
– May block or stimulate transcription
• Prokaryotic organisms regulate gene expression
in response to their environment
• Eukaryotic cells regulate gene expression to
maintain homeostasis in the organism
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Regulatory Proteins
• Gene expression is often controlled by
regulatory proteins binding to specific DNA
sequences
– Regulatory proteins gain access to the bases
of DNA at the major groove
– Regulatory proteins possess DNA-binding
motifs
3
DNA-binding motifs
• Regions of regulatory proteins which bind
to DNA
– Helix-turn-helix motif
• Homeodomain motif
– Zinc finger motif
– Leucine zipper motif
4
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The Helix-Turn-Helix Motif
α Helix
(Recognition helix)
Turn
The Leucine Zipper Motif
Turn
Zipper region
α Helix
α Helix
3.4 nm
90°
a.
b.
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Prokaryotic regulation
• Control of transcription initiation
– Positive control – increases frequency of
initiation of transcription
• Activators enhance binding of RNA polymerase to
promoter
• Effector molecules can enhance or decrease
– Negative control – decreases frequency
• Repressors bind to operators in DNA
• Allosterically regulated
• Respond to effector molecules – enhance or
abolish binding to DNA
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• Prokaryotic cells often respond to their
environment by changes in gene
expression
• Genes involved in the same metabolic
pathway are organized in operons
• Induction – enzymes for a certain pathway
are produced in response to a substrate
• Repression – capable of making an
enzyme but does not
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Glucose repression
• Preferential use of glucose in the presence of
other sugars
– Mechanism involves activator protein that stimulates
transcription
– Catabolic activator protein (CAP) is an allosteric
protein with cAMP as effector
– Level of cAMP in cells is reduced in the presence of
glucose so that no stimulation of transcription from
CAP-responsive operons takes place
• Inducer exclusion – presence of glucose inhibits
the transport of lactose into the cell
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Eukaryotic Regulation
• Control of transcription more complex
• Major differences from prokaryotes
– Eukaryotes have DNA organized into
chromatin
• Complicates protein-DNA interaction
– Eukaryotic transcription occurs in nucleus
• Amount of DNA involved in regulating
eukaryotic genes much larger
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Transcription factors
• General transcription factors
– Necessary for the assembly of a transcription
apparatus and recruitment of RNA
polymerase II to a promoter
– TFIID recognizes TATA box sequences
• Specific transcription factors
– Increase the level of transcription in certain
cell types or in response to signals
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• Promoters form the binding sites for
general transcription factors
– Mediate the binding of RNA polymerase II to
the promoter
• Enhancers are the binding site of the
specific transcription factors
– DNA bends to form loop to position enhancer
closer to promoter
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• Coactivators and mediators are also
required for the function of transcription
factors
– Bind to transcription factors and bind to other
parts of the transcription apparatus
– Mediators essential to some but not all
transcription factors
– Number of coactivators is small because used
with multiple transcription factors
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Transcription complex
• Few general principles
• Nearly every eukaryotic gene represents a
unique case
• Great flexibility to respond to many signals
• Virtually all genes that are transcribed by
RNA polymerase II need the same suite of
general factors to assemble an initiation
complex
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Eukaryotic chromatin structure
• Structure is directly related to the control
of gene expression
• DNA wound around histone proteins to
form nucleosomes
• Nucleosomes may block access to
promoter
• Histones can be modified to result in
greater condensation
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• Methylation once thought to play a major role in
gene regulation
– Many inactive mammalian genes are methylated
– Lesser role in blocking accidental transcription of
genes turned off
• Histones can be modified
– Correlated with active versus inactive regions of
chromatin
– Can be methylated – found in inactive regions
– Can be acetylated – found in active regions
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• Some coactivators have been shown to be
histone acetylases
• Transcription is increased by removing
higher order chromatin structure that
would prevent transcription
• “Histone code” postulated to underlie the
control of chromatin structure
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• Chromatin-remodeling complexes
– Large complex of proteins
– Modify histones and DNA
– Also change chromatin structure
• ATP-dependent chromatin remodeling factors
– Function as molecular motors
– Catalyze 4 different changes in DNA/histone binding
– Make DNA more accessible to regulatory proteins
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ATP
ADP+ Pi
ATP -dependent
remodeling factor
1. Nucleosome sliding
2. Remodeled nucleosome
3. Nucleosome displacement
4. Histone replacement
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Posttranscriptional Regulation
• Control of gene expression usually involves
the control of transcription initiation
• Gene expression can be controlled after
transcription with
– Small RNAs
• miRNA and siRNA
– Alternative splicing
– RNA editing
– mRNA degradation
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Micro RNA or miRNA
• Production of a functional miRNA begins in the
nucleus
• Ends in the cytoplasm with a ~22 nt RNA that
functions to repress gene expression
• miRNA loaded into RNA induced silencing
complex (RISC)
• RISC is targeted to repress the expression of
genes based on sequence complementarity to
the miRNA
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RNA Polymerase II
microRNA gene
Pri-microRNA
Nucleus
Pre-microRNA
Drosha
Exportin 5
Cytoplasm
Dicer
Mature miRNA
RISC
mRNA
RISC
mRNA cleavage
mRNA
RISC
Inhibition of translation
RISC
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siRNA
• RNA interference involves the production
of siRNAs
• Production similar to miRNAs but siRNAs
arise from long double-stranded RNA
• Dicer cuts yield multiple siRNAs to load
into RISC
• Target mRNA is cleaved
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miRNA or siRNA?
• Biogenesis of both miRNA and siRNA
involves cleavage by Dicer and
incorporation into a RISC complex
• Main difference is target
– miRNA repress genes different from their
origin
– Endogenous siRNAs tend to repress genes
they were derived from
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Exogenous dsRNA, transposon, virus
Repeated cutting
by dicer
siRNAs
siRNA
in RISC
Ago
RISC
+
Ago
RISC
mRNA
Cleavage of target mRNA
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Alternative splicing
• Introns are spliced out of pre-mRNAs to
produce the mature mRNA
• Tissue-specific alternative splicing
• Same gene makes calcitonin in the thyroid
and calcitonin-gene related peptide (CGRP)
in the hypothalamus
• Determined by tissue-specific factors that
regulate the processing of the primary
transcript
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RNA editing
• Creates mature mRNA that are not truly
encoded by the genome
• Involves chemical modification of a base
to change its base-pairing properties
• Apolipoprotein B exists in 2 isoforms
– One isoform is produced by editing the
mRNA to create a stop codon
– This RNA editing is tissue-specific
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• Initiation of translation can be controlled
– Ferritin mRNA only translated if iron present
• Mature mRNA molecules have various
half-lives depending on the gene and the
location (tissue) of expression
– Target near poly-A tail can cause loss of the
tail and destabilization
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RNA polymerase II
DNA
3´
5´
Primary RNA transcript
1. Initiation of
transcription
Most control of
gene expression
is achieved
by regulating
the frequency
of transcription
initiation.
2. RNA splicing
Cut
Gene expression
intron
3´ poly-A tail
5´ cap
Exons
Introns
can be controlled
by altering the
rate of splicing in
eukaryotes.
Alternative splicing
can produce
multiple mRNAs
from one gene.
Mature RNA transcipt
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Large
subunit
3´ poly-A tail
Nuclear
pore
mRNA
Small
subunit
3´
5´
3. Passage through
the nuclear
membrane
Gene expression
can be regulated
by controlling
access to or
efficiency of
5´ cap
transport channels.
4. Protein synthesis
Many proteins take
part in the
translation process,
and regulation
of the availability
of any of them alters
the rate of gene
expression by
speeding or slowing
protein synthesis.
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P
P
Completed 6. Posttranslational
polypeptide
modification
chain
Phosphorylation
or other chemical
modifications can
alter the activity
of a protein after
it is produced.
RISC
5. RNA interference
Gene expression
is regulated by
small RNAs. Protein
complexes
containing siRNA
and miRNA target
specific mRNAs for
destruction or inhibit
their translation.
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Protein Degradation
• Proteins are produced and degraded
continually in the cell
• Lysosomes house proteases for
nonspecific protein digestion
• Proteins marked specifically for
destruction with ubiquitin
• Degradation of proteins marked with
ubiquitin occurs at the proteasome
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