Transcript Freeman 1e: How we got there - EvergreenStateCollege-Home

Control of Gene Expression in Bacteria
Gene Regulation and Information Flow: an overview
Metabolizing Lactose—A Model System: multiple genes, classes
of mutants; the lac operon model and the discovery of the
repressor (Jacob and Monod)
Catabolite Repression and Positive Control: How Does Glucose
Influence Formation of the CAP-cAMP Complex?
The Operator and the Repressor—an Introduction to DNABinding Proteins: Finding the Operator; DNA Binding via the
Helix-Turn-Helix Motif
How Does the Inducer Change the Repressor’s Affinity for DNA?
17.1 Gene Regulation and Information Flow
• Escherichia coli has served as an excellent model
organism for the study of prokaryotic gene regulation
because, like most bacteria, it can use a wide array of
carbohydrates to supply carbon and energy.
•Producing all the enzymes required to process all the
various carbohydrates all the time would waste energy. It
is logical to predict that the enzymes E. coli produces
match the sugars that are available at a given time.
•Efficient use of resources, via tight control over gene
expression, is critical for E. coli's survival.
• Glucose is the preferred carbon source for E.
coli. Lactose is used only when glucose is
depleted.
•E. coli produces high levels of b-galactosidase,
the enzyme that cleaves lactose to glucose +
galactose, only when lactose is present in the
environment. Thus, lactose acts as an inducer—a
molecule that stimulates the expression of a
specific gene.
•Jacques Monod found that b-galactosidase is not
expressed in E. coli cells grown in medium
containing glucose or glucose + lactose but only in
medium containing lactose and no glucose
Master plates containing medium with many sugars were
replica-plated to medium with lactose as the only sugar to
screen for colonies that could not grow on lactose
• Indicator plates allow mutants with metabolic deficiencies to be observed
directly. Colonies grown on lactose were sprayed with ONPG (o-nitrophenol-bD-galactoside), an indicator with a structure similar to that of lactose. When bgalactosidase breaks down ONPG, the intensely yellow compound onitrophenol is produced, turning colonies bright yellow.
Indicator plates allow mutants with metabolic deficiencies to be
observed directly. Colonies grown on lactose were sprayed with
ONPG (o-nitrophenol-β-D-galactoside), an indicator with a structure
similar to that of lactose. When β-galactosidase breaks down ONPG,
the intensely yellow compound o-nitrophenol is produced, turning
colonies bright yellow. Three classes of mutants were found:
The lac Operon
• Jacob and Monod coined the term operon for a set of
coordinately regulated bacterial genes that are transcribed
together into one mRNA, usually encoding proteins that work
together.
•The group of genes involved in lactose metabolism was termed the
lac operon.
•The lac operon genes are expressed on a single polycistronic
mRNA, and their expression is regulated by a single promoter.
•The repressor does not physically block RNA polymerase from
contacting the promoter but instead prevents transcription
initiation by keeping RNA polymerase from unwinding the
DNA helix.
The Impact of the lac Operon Model
• The lac operon model introduced the idea that gene
expression is regulated by physical contact between
regulatory proteins and regulatory sites within the DNA.
•Negative control occurs when something must be taken
away for transcription to occur.
•The lac operon repressor exerts negative control over
three protein-coding genes by binding to the operator
site in DNA near the promoter. For transcription to occur,
an inducer molecule (a derivative of lactose) must bind to
the repressor, causing it to release from the operator
Superimposed positive control:
Inhibition of a metabolic pathway by its breakdown products
(end-product inhibition) is called catabolite repression (e.g.,
glucose inhibiting the lactose operon).
• When little glucose is available and less ATP is produced,
a derivative of ATP called cyclic AMP (cAMP) is produced.
•cAMP allows activation of expression of the lac operon
through binding to the catabolite activator protein (CAP),
which binds a DNA sequence called the CAP binding site
located just upstream of the lac promoter.
•Binding by the positive regulator CAP strengthens the lac
promoter to increase expression.
Finding the
operator site
in the DNA:
DNA footprinting
is used to identify
DNA sequences
that are bound by
regulatory
proteins.
•The section of a helix-turn-helix regulatory protein that binds inside
the DNA major groove is called the recognition sequence; it
recognizes and interacts with the specific nitrogenous bases there.
The active lac repressor is a tetramer of four LacI+ monomers, each of
which can bind to an operator sequence to block the opening of the double
helix for transcription. One tetramer can bind two operator sequences and
cause the DNA between the operators to “kink” or “loop”
When the repressor interacts with the inducer (either lactose or IPTG, an
analog of lactose), the inducer binds to a central region of the repressor
and induces a change in the shape of the repressor tetramer, making it
release the DNA.
Vibrio cholerae – a bacterial pathogen
Regulation of toxin gene expression in cholera: pg. 378-9
Note: this group of genes is not in most Vibrio cholera bacteria; it is brought in
on a particular temperate phage that becomes a prophage. A second temperate
phage, this one of the filamentous family, is responsible for the pilus that binds
the bacteria in place in the small intestine as its toxin stimulates human adenyl
cyclase, leading to massive secretion of chloride ions and thus water, producing
the potentially-lethal severe diarrhea.
Eukaryotic Regulation:
•There are 4 primary differences
between gene expression in
bacteria and eukaryotes:
•(1) Packaging of DNA
(chromatin structure) in
eukaryotes;
•(2) splicing of mRNA in
eukaryotes;
•(3) complexity of transcriptional
control in eukaryotes;
•(4) coordinated expression
through operons in bacteria.
Alternate mRNA splicing in different tissue types:
•At least 35% of human genes undergo alternative splicing. Although we have
only around 40,000 genes, it is anticipated that we express between 100,000 and
1 million different protein products.
Experiment carried out in reticulocytes
(young red blood cells)
Ovalbumin gene
β-globin gene (hemoglobin subunit)
•Histone acetyl transferases (HATs) add negatively charged acetyls (acetylation) or
methyls (methylation) to histones. This decondenses the chromatin and allows gene
expression. Histone deacetylases (HDACs) remove the acetyl groups from histones
to allow chromatin condensation and turn off gene expression.
p53 – Guardian of the Genome and Tumor Suppressor
The signal transducers
and activators of
transcription (STATs)
are an excellent example
of post- translational
regulation via protein
phosphorylation
The gene encoding the splicing factor SF2/ASF is a proto-oncogene
Rotem Karni, Elisa de Stanchina, Scott W Lowe, Rahul Sinha, David Mu & Adrian R
Krainer [email protected] Nature Structural & Molecular Biology - 14, 185 - 193 (2007)
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Alternative splicing modulates the expression of many oncogene and
tumor-suppressor isoforms. We have tested whether some alternative
splicing factors are involved in cancer. We found that the splicing factor
SF2/ASF is upregulated in various human tumors, in part due to
amplification of its gene, SFRS1. Moreover, slight overexpression of
SF2/ASF is sufficient to transform immortal rodent fibroblasts, which
form sarcomas in nude mice.
We further show that SF2/ASF controls alternative splicing of the tumor
suppressor BIN1 and the kinases MNK2 and S6K1. The resulting BIN1
isoforms lack tumor-suppressor activity; an isoform of MNK2 promotes
MAP kinase–independent eIF4E phosphorylation; and an unusual
oncogenic isoform of S6K1 recapitulates the transforming activity of
SF2/ASF. Knockdown of either SF2/ASF or isoform-2 of S6K1 is sufficient
to reverse transformation caused by the overexpression of SF2/ASF in
vitro and in vivo.
Thus, SF2/ASF can act as an oncoprotein and is a potential target for
cancer therapy.