Control%20of%20Gene%20Expression%20in%20Bacteria

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• Control of Gene Expression in Bacteria
• Gene Regulation and Information Flow an overview
• Metabolizing LactoseA 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 Repressoran Introduction to
  DNA-Binding Proteins Finding the Operator DNA
  Binding via the Helix-Turn-Helix Motif
• How Does the Inducer Change the Repressors
  Affinity for DNA?

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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.

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• 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 inducera
  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

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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
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• Indicator plates allow mutants with metabolic
  deficiencies to be observed directly. Colonies
  grown on lactose were sprayed with ONPG
  (o-nitrophenol-b-D-galactoside), an indicator
  with a structure similar to that of lactose. When
  b-galactosidase breaks down ONPG, the intensely
  yellow compound o-nitrophenol is produced,
  turning colonies bright yellow.

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Indicator plates allow mutants with metabolic
deficiencies to be observed directly. Colonies
grown on lactose were sprayed with ONPG
(o-nitrophenol-ß-D-galactoside), an indicator
with a structure similar to that of lactose. When
ß-galactosidase breaks down ONPG, the intensely
yellow compound o-nitrophenol is produced,
turning colonies bright yellow. Three classes of
mutants were found
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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.

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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

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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).
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• 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.

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Finding the operator site in the DNA DNA
footprinting is used to identify DNA sequences
that are bound by regulatory proteins.
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• 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.

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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.
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Vibrio cholerae a bacterial pathogen
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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.
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• 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.

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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.

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Experiment carried out in reticulocytes
(young red blood cells)
Ovalbumin gene ß-globin gene
(hemoglobin subunit)
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• 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.

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p53 Guardian of the Genome and Tumor
Suppressor
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The signal transducers and activators of
transcription (STATs) are an excellent example of
post- translational regulation via protein
phosphorylation
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The gene encoding the splicing factor SF2/ASF is
a proto-oncogeneRotem Karni, Elisa de Stanchina,
Scott W Lowe, Rahul Sinha, David Mu  Adrian R
Krainer krainer_at_cshl.edu Nature Structural
Molecular Biology - 14, 185 - 193 (2007)

• 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 kinaseindependent
  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.