Transcript BIOL 112 – Principles of Zoology

I. Overview of Eukaryotic gene
regulation
Mechanisms similar to
those found in bacteriamost genes controlled
at the transcriptional
level
Much more complex
than prokaryotic
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chromatin
TFs
Enhancers
Activators
A. Prokaryotes vs. Eukaryotes
 In eukaryotes, one mRNA = one protein.
(in bacteria, one mRNA can be polycistronic, or code
for several proteins).
 DNA in eukaryotes forms a stable, compacted
complex with histones. In bacteria, the chromatin is
not in a permanently condensed state.
 Eukaryotic DNA contains large regions of repetitive
DNA, whilst bacterial DNA rarely contains any "extra"
DNA.
 Eukaryotic genes are divided into exons and introns;
in bacteria, genes are almost never divided.
 In eukaryotes, mRNA is synthesized in the nucleus
and then processed and exported to the cytoplasm; in
bacteria, transcription and translation can take place
simultaneously off the same piece of DNA.
B. Eukaryote gene expression
is regulated at 6 levels:
1. Transcription
2. RNA processing
3. mRNA transport
4. mRNA translation
5. mRNA degradation
6. Protein degradation
II. Transcriptional Control
A. Control factors
1) cis-acting “next to” elements
Promoter region: TATA box (-30), CAAT box
80) GC box (-110)
Alternate promoters
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The level of transcription initiation can vary between
alternative promoters
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the translation efficiency of mRNA isoforms with
different leader exons can differ
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alternative promoters can have different tissue
specificity and react differently to some signals
Enhancers & Silencers far away from promoter
2) trans-acting “across from” factors
Transcription factors
Activators, Coactivators
(-
Control factors continued:
3) DNA methylation (add methyl to C)
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Occurs at 5’ position, usually in CG doublets
5’-mCpG-3’
Inverse relationship between degree of methylation
and degree of expression
Not a general mechanism in eukaryotes
Transcriptionally active genes possess
significantly lower levels of methylated DNA than
inactive genes.
A gene for methylation is essential for development in
mice (turning off a gene also can be important).
Methylation results in a human disease called fragile
X syndrome; FMR-1 gene is silenced by methylation.
Control factors continued:
4) Chromatin conformation (remodelling)
a. Antirepressors & nucleosome positioning.
b. Histone acetylation – (acetyl groups on lysines),
histone acetyltransferase enzyme catalyzes the
addition of lysine, targeted to genes by specific
TFs.
c. Heterochromatin – highly condensed,
transcriptionally inert (off).
B. Eukaryotic Promoters
Usually located within 100 bp upstream
Usually contains TATA box (25 – 30) bases upstream from start
point, additional elements:
 CAAT box
 GC box
 Recognized byRNA Pol II (transcribes mRNA)
 Require the binding of several protein factors to initiate
transcription (DNA binding domains on TFs – ‘motifs’)
 May be positively or negatively regulated
C. Transcription Factors –the
transcription complex
1) TFIIA, TFIIB,
TFIID, TFIIE,
TFIIH
2) TATA binding
protein (TBP)
3) TBP associated
factors (TAFs)
Assembly of the basal
transcription apparatus involves stepwise binding
of various transcription
factor proteins.
These trans-acting
proteins are required for
RNA pol II to initiate
transcription.
Commitment Stage &
Clearance Stage…
Activators are required to
bring about normal levels
of transcription
Enhancers
Cis regulators that interact
with regulatory proteins &
TFs to increase the
efficiency of transcription
initiation.
Silencers – cis-acting,
bound by repressors, or
cause the chromatin to
condense and become
inactive.
Activators - Proteins that
function by contacting basal
transcription factors and
stimulating the assembly of
pre-initiation complexes at
promoters.
D. An example of transcriptional control:
Galactose metabolism in yeast
 GAL1, GAL7, GAL10 genes… products
required for conversion of galactose into
glucose
Closely linked genes, but monocistronic mRNAs synthesized
These are only transcribed when galactose is present…
Galactose metabolizing
pathway of yeast.
Controlling GAL
GAL80 encodes a protein that negatively regulates
transcription. The repressor protein binds to an
Activator protein, rendering it inactive.
GAL4 encodes an activator w/zinc finger motif that
activates transcription of the three GAL genes
individually.
Galactose = Inducer, that binds to Gal80, causing
it to release Gal4
 Although this looks similar to Lac Operon, there
are different molecular mechanisms…
Two trans-acting
genes (GAL4 and
GAL80) and one
upstream cis-acting
locus (UAS) work to
regulate
galactokinase
synthesis.
Activation model of GAL genes in yeast.
III. Post-transcriptional control
A. Alternative splicing - Some messages undergo
alternate splicing depending on what tissue they
are located in. The regulation is at the level of
snRNP production.
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Some pre-mRNAs can be spliced in more than one way,
producing 2+ alternative mRNA’s
Can introduce stop codons or change the reading frame
Controlled by RNA binding splicing factors that commit
splicing in a particular way
Alternative polyadenylation and splicing of the human CACL gene in
thyroid and neuronal cells.
Calcitonin
gene-related
peptide
Post-transcriptional control cont.
B.
The stability of a class of mRNA can be controlled.
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C.
Some short-lived mRNAs have multiple copies of the sequence
AUUUA which may act as a target for degradation.
the hormone prolactin enhances the stability of the mRNA for the
milk protein casein
high levels of iron decrease the stability of the mRNA for the
receptor that brings iron into cells
RNA interference – poorly understood, but appears to be
widespread in fungi, plants and animals as a regulatory
mechanism
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miRNAs & siRNAS (small RNA molecules) pair with proteins to
form an RNA-induced silencing complex (RISC)
RISC pairs w/complentary base sequences of specific mRNAs and
causes:
1)
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3)
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Cleavage of mRNA
Inhibition of translation
Transcriptional silencing
Degradation of mRNA