Freeman 1e: How we got there

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Signal transduction
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Signal transduction and cell division
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Signal transduction
in prokaryotes
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Regulation of Enzyme Activity 
Feedback Inhibition
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• An allosteric enzyme has two binding sites, the
  active site, where the substrate binds, and the
  allosteric site, where the inhibitor (called an
  effector) binds reversibly

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glucose
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glucose
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These sugars must be proceeded before entering in
glycolysis
glucose
arabinose
lactose
galactose
maltose
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Induction of lac operon (negative regulation)
(beta galactosidase)
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Expression of arginine biosynthetic pathway
(Operon)
(Arginine)
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trp arg
aporrepresor correpresor
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Maltose operon regulation (positive)
(Absence of inducer - maltose)
(maltose)
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Constitutive mutant (Oc)
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Constitutive mutant (I-)
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mutant non inducible (Is e Iq)
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Lac operator
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IPTG
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An Overview of Gene Regulation
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Negative Control of Transcription Repression and
Induction
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• This transcriptional regulation involves
  allosteric regulatory proteins that bind to DNA.
  For negative control of transcription, the
  regulatory molecule is called a repressor protein
  and it functions by inhibiting mRNA synthesis.

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Positive Control of Transcription

• Positive control of transcription is implemented
  by regulators called activator proteins. They
  bind to activator-binding sites on the DNA and
  stimulate transcription. As in repressors,
  activator protein activity is modified by
  effectors.

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• For positive control of enzyme induction, the
  effector promotes the binding of the activator
  protein and thus stimulates transcription

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Operon

• bacterial operons allows the bacteria to
  regulate its transcription/translation machinery
  using the environment
• operons participate in metabolic control
• turned on or off based on the metabolic
  requirements of the bacteria
• operon a functioning unit of genomic DNA
  containing a cluster of genes under the control
  of a single regulatory signal or promoter. The
  genes are transcribed together into an mRNA
  strand and. The result of this is that the genes
  contained in the operon are either expressed
  together or not at all. Several genes must be
  both co-transcribed and co-regulated to define an
  operon
• e.g. trp-operon, lac-operon
• promoter controls transcription of multiple
  downstream genes that code for enzymes that
  synthesize a specific metabolic component (e.g.
  amino acid)
• these downstream genes are called a transcription
  unit
• also contains an operator region on/off switch
  that ultimately controls the promoter and the
  downstream genes

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resumen
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In eukaryotes nuclear receptors are always bound
to operator response element

• No inducer ? recruits repressors
• With inducer ? recruits activators

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What does bacterium have to do
?
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diauxie
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Global Regulatory Mechanisms  
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• Global control systems regulate the expression
  of many genes simultaneously. Catabolite
  repression is a global control system, and it
  helps cells make the most efficient use of carbon
  sources.

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• The phenomenon of catabolite repression has been
  observed in operons other than the lac operon. In
  fact, the expression of many operons that play
  roles in sugar metabolism and amino acid
  metabolism is affected by the presence of
  glucose. Examples are the arabinose (ara)
  operon the maltose (mal) operon and, the
  histidine utilization (hut) operon. This is a
  GLOBAL CONTROL MECHANISM.
• The global nature of the catabolite repression
  phenomenon makes sense. As long as glucose is
  present, it will be the preferred substrate for
  growth, so there will be no need for any of the
  other substances to be used and, consequently,
  for their operons to be expressed.
• Catabolite repression is mediated through the
  effects that glucose transport into the cell has
  on the internal concentration of cyclic AMP
  (cAMP). The following "cartoon" shows how this
  works
• If glucose is abundant in the growth medium it
  will be transported in to the cell by the action
  the glucose transport system. As it is being
  transported, glucose is phosphorylated with the
  phosphate group being donated by a component of
  the transport system.The same component also
  activates the enzyme, adenylate cyclase. As long
  as the component is participating in glucose
  transport, it is not able to activate adenylate
  cyclase.The result is that as glucose is
  transported into the cell, the concentration of
  cAMP falls (because adenylate cyclase is not
  being activated to synthesize any more).
• If there is little or no glucose in the growth
  medium, the glucose transport system is not
  operational. The phosphate donor component is now
  free to activate aadenylate cyclase.The result
  is that in the absence of glucose, the
  concentration of cAMP rises.
• Thus there is an inverse relationship between
  glucose and cAMP. As one rises, the other
  falls.

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cAMP and CAP(catabolite activator protein)
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High glucose ? low cAMP ? no CAP binding to DNA
? no activation
Low glucose ? high cAMP ? cAMP-CAP binding to DNA
? activation
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• The lac operon is under the control of
  catabolite repression as well as its own specific
  negative regulatory system

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The Stringent Response

• is a global control mechanism triggered by amino
  acid starvation.

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The alarmones ppGpp and pppGpp are produced by
RelA, a protein that monitors ribosome activity.
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ppGpp
??-??-???-??-??
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Other Global Control Networks

• Cells can control several regulons by employing
  alternative sigma factors. Alternative signal
  factors in Escherichia coli are shown in Table
  8.2.

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Sigma factor binds DNA
RNAP holoenzyme binds Sigma factor DNA complex
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• These recognize only certain promoters and thus
  allow transcription of a select category of
  genes. Other global signals include cold and heat
  shock proteins that function to help the cell
  overcome temperature stress.

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• Table 8.1 shows examples of global control
  systems known in Escherichia coli.

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Sigma38 changes pol to respond to this
regulation without repressors or activators
Normal Transcription
Osm promoters contain poised polymerases
Osmotic shock
potassium glutamate accumulation, inhibited s70
transcription
Neutral osmolytes restore sigma70 function
Poised polymerases begin transcription
s38 polymerase produces neutral osmolytes
key unresolved question How does the weak acid
salt induce the poised RNAP to escape?
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CIRCE ???????
Heat shock
N N N N N N N N
N C-G T-A C-G A-T C-G
G-C A-T T-A T-A NNNNN
NNNNN NNNNN NNNNN A-T A-T T-A
C-G G-C T-A G-C A-T
G-C N N N N N N N N
N
N N N N N N N N
N C-G T-A C-G A-T C-G
G-C A-T T-A T-A NNNNN NNNNN
HrcA
TTAGCACTC---NNNNNNNNN---GAGTGCTAA
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9
9
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CIRCE ???????
37 C
??????????????? ?????
44 C
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Quorum Sensing

• Quorum sensing (Figure 8.22) allows cells to
  survey their environment for cells of their own
  kind and involves the sharing of specific small
  molecules. Once a sufficient concentration of the
  signaling molecule is present, specific gene
  expression is triggered.

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Other Mechanisms of RegulationAttenuation
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• Attenuation is a mechanism whereby gene
  expression (typically at the level of
  transcription) is controlled after initiation of
  RNA synthesis (Figure 8.25).

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• Attenuation mechanisms involve a coupling of
  transcription and translation and can therefore
  occur only in prokaryotes.

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• Protein splicing (Figure 8.7) is a form of
  posttranslational modification.

Topoisomerase II (GyrA) in Mycobacterium
leprae (Lyprosae disease)
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Signal Transduction and Two-Component Regulatory
Systems
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• Signal transduction systems transmit
  environmental signals to the cell.

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• In prokaryotes, signal transduction typically
  involves two-component regulatory systems (Figure
  8.26), which include a membrane-integrated sensor
  kinase protein and a cytoplasmic response
  regulator protein.
• The activity of the response regulator depends on
  its state of phosphorylation.

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• Table 8.3 shows examples of two-component
  regulatory systems that regulate transcription in
  Escherichia coli.

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Regulation of Chemotaxis

• Chemotaxis is under complex regulation involving
  signal transduction in which regulation occurs in
  the activity of the proteins involved rather than
  in their synthesis (Figure 8.27).

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MCP methyl accepting chemotaxis proteins
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• Adaptation by methylation allows the system to
  reset itself to the continued presence of a
  signal.

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RNA Regulation and Riboswitches

• RNA regulation is a rapidly expanding area in
  both prokaryotic and eukaryotic molecular biology.

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• In Escherichia coli, for example, a number of
  small RNAs (sRNAs) have been found to regulate
  various aspects of cell physiology by binding to
  other RNAs or even to small molecules.

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• One mechanisms for sRNA activity is found in the
  signal recognition particle. A second mechanism
  is the binding of the sRNA to an mRNA by
  complementary base pairing (Figure 8.28a).

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• A unique form of small RNAs are the
  riboswitches. These are mRNAs that contain
  sequences upstream of the coding sequences that
  can bind small molecules (Figure 8.28b).

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Metabolite binding effects secondary structure of
RNA
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Covalent Modification of Enzymes

• Covalent modification is a regulatory mechanism
  for changing the activity of an enzyme. Enzymes
  regulated in this way can be reversibly modified.
  One type of modification is adenylylation (the
  addition of AMP) (Figure 8.6).
• Others are phosphorylation and methylations

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Regulation of glutamine synthetase by
adenylation 1. GS inhibition by several a. a. and
compounds involved in nucleotide metabolism 2. GS
(12 identical subunits) is also inhibited by
covalent modification - adenylation
Nitrogen rich medium (Lot of glutamine available)
Nitrogen poor medium
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DNA-Binding Proteins and Regulation of
Transcription by Negative And Positive Control

• Certain proteins can bind to DNA because of
  interactions between specific domains of the
  proteins and specific regions of the DNA molecule
  (Figure 8.8).

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DNA Binding Proteins
homodimers
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• In most cases, the interactions are
  sequence-specific. Proteins that bind to nucleic
  acid may be enzymes that use nucleic acid as
  substrates, or they may be regulatory proteins
  that affect gene expression.

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• Several classes of protein domains are critical
  for proper binding of these proteins to DNA. One
  of these is called the helix-turn-helix motif
  (Figure 8.9).

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Lac and trp repressors and lambda repressor (gt250)
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Cysteine (C) and histidine (H)

• Another domain is the zinc finger, a protein
  that binds a zinc ion (eukaryotes).