Biol 568 Advanced Topics in Molecular Genetics

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Biol 568Advanced Topics in Molecular Genetics
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Ch 11 Regulatory Circuits
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• Regulatory circuits
• Positive and negative regulators
• Small molecule effectors
• Regulators
• Expression
• Transcription
• Translation

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Regulatory circuits

• In general
• A gene is controlled by a regulator
• The Regulator
• Interacts with specific DNA or RNA sequences or
  structures
• Turns off or on the expression/translation
• Can be controlled by other regulators

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Regulatory circuits

• Regulatory circuits
• One regulator is required for expression or
  regulation of a (several) successive regulator (s)

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Regulatory circuits
Figure 11.2
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Regulatory circuits

• Protein regulators
• principle of allostery
• RNA regulators
• changes in secondary structure

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Regulatory circuits
Fig. 11.1
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• Regulatory circuits
• Positive and negative regulators
• Small molecule effectors
• Regulators
• Expression
• Transcription
• Translation

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Positive and negative regulators

• Positive or negative regulators
• Defined by the response of the operon when no
  regulator is present
• Negative Regulation
• The operon is expressed in the absence of
  regulator
• Positive regulation
• The operon is suppressed

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Negative regulators

• Negative regulation a fail safe mechanism
• How might a system of negative regulation have
  evolved?
• The operon is first expressed constitutively
• The cells in which the expression is somehow
  interrupted gain a selective advantage in the
  overall population

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Positive regulators

• Positive regulation counterpart of the negative
  regulation
• The regulator is required for activation of
  transcritption
• sigma factor (s)

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Positive Regulator

• Interact with DNA and/or RNA polymerase to
  activate transcription
• Activator
• A positive regulator protein that responds to a
  small molecule

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Positive and negative regulators

• How has the positive regulation evolved?
• The activator part of the overall transcription
  complex, later restricted to one particular set
  or a singular gene

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Positive and negative regulators

• Operons
• Inducible
• function in the presence of the small-molecule
  inducer
• Repressible
• function only in the absence of the small
  molecule corepressor
• Derepressed Induced
• Super-repressed Uninduced

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Positive and negative regulators
Figure 11.3
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Positive and negative regulators

• When the interaction of a small molecule with
  the regulator leads to activation of expression
  this molecule is called inducer
• If the inducer
• Inactivates the repressor - ve control
• Activates the activator ve control
• Both systems are inducible systems

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Positive and negative regulators

• When the interaction of a small molecule with the
  regulator leads to inactivation of transcription
  is called corepressor
• The corepressor
• Activates the repressor - ve control
• Inactivate the activator ve control
• Both systems are repressible systems

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Positive and negative regulators

• Regulation of the repressor
• Allosteric changes
• Other
• Oxidation
• OxyR ve regulation
• OxyR activator
• induced by hyrogen peroxidase
• Phosphorylation

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Negative Regulators

• Lac Operon
• Negative regulation
• Repressor is inactivated by the inducer
• Inducer Lactose
• How is the inducer regulated?

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Glucose Repression

• Glucose is used as the carbon source of
  preference
• Prevents the uptake of alternative carbon sources
  
• Exclusion of alternative carbon sources prevents
  expression of the operons coding for enzymes that
  metabolize the alternative sources.

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Glucose Repression

• Inhibition of alternative carbon sources caused
  by the uptake of glucose
• Glucose repression
• Inducer exclusion

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Glucose Repression

• Figure 11.4
• Glucose is imported by phosphoenolpyruvate
• PTS
• glycose phosphotransferase system
• Lac Permease is inactivated
• Lac is operon turned off

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Positive Regulation

• CRP/CAP activator
• CRP positive regulator
• Activated by a small molecule cAMP
• CRP activator

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CRP activator

• Binds to promoters to activate transcription
• Several promoters require binding of ancillary
  proteins for transcription
• Common in promoters with poor -35, -10 consensus
  sites
• Activator for a large set of operons in E coli.
• Active only in the presence of cAMP.

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cAMP

• 5 and 3 positions in the sugar ring connected
  via a phosphate group
• Activates CRP at many promoters

Figure 11.5
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Positive control by CRP
Figure 11.6
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Positive control by CRP

• Positive control
• Inducible system
• cAMP present -gt Cap is activated
• Txn On
• cAMP absent/reduced -gt CAP is inactive
• Txn Off

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Positive control by CRP

• CRP
• Dimer, two identical subunits activated by a
  single cAMP molecule
• Binds to DNA
• CRP-cAMP-DNA complexes can be isolated at each
  promoter
• CRP monomer contains a DNA binding region and a
  transcriptional activating region

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Consensus sequence for CRP
Figure 11.7
Two domains A well conserved pentamer
(-22) TGTGA An inverted sequence of pentamer
TCANA
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CRP

• Figure 11.8 CRP binds to different sites

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• lac operon
• CRP site at -41
• RNA and CRP may be in contact with each other
• CRP interacts with the same strand of DNA and
  from the same side of DNA that the RNA pol. does

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• ara operon
• CRP site is further upstream at -92
• gal operon
• CRP site at -41, probably one monomer binds
• CRP site within the RNA protected region

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• The majority of CRP binding sites are either
• Lac-like ( -61) Class I
• or
• Gal-like ( -41) Class II
• CRP interacts with a subunit of RNA pol

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CRP and RNA pol Interaction

• Clas I promoters
• Increases the rate of transitional binding to
  form a closed complex
• Stabilizes the RNA pol binding
• ClassII promoters
• CRP binding site located further upstream
• Facilitates the transition form a closed complex
  to an open complex

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DNA bending assays
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CRP bends DNA

• Figure 10.11
• Results analyzed by plotting mobility
• 90o bend between the two repeats
• Introduces a sharp change in DNA structure
• May bring CRP close to RNA for interaction

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Regulation of lac operon

• Glucose present
• Lac permease is inhibited
• Inducer is excluded
• cAMP is inhibited
• CRP is inactive
• The operon is repressed

• Lactose present
• Repressor is inactivated
• Repression is released
• cAMP activates CRP
• Txn is activated
• The operon is expressed

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• Regulatory circuits
• Positive and negative regulators
• Small molecule effectors
• Regulators
• Expression
• Transcription
• Translation

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The stringency response

• Stringent conditions
• Insufficient supply for the aa required for
  protein synthesis
• Stringent response
• Reduction in the rRNA synthesis
• mRNA synthesis decreased at least 3X
• Increase in the rate of protein degradation

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Stirngent response

• Accumulation of two nucleotides
• ppGpp and pppGpp
• small effectors
• bind to target proteins to alter their activities

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Stringent Factor

• Ribosomal proteins phosphorylate pppGpp to
  ppGpp

Figure 11.11
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• Rel A catalyzator for (p)ppGpp synthesis
• spoT degrades (p)ppGpp
• ( half life 20sec)
• Dephosphorylation of pppGpp is the most common
  pathway ( EF-Tu, EF-G dephosporylators)

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RelA responds to stringency
Figure 11.12
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• pppGpp -gt ppGpp
• ppGpp effector of the stringent response

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• Normal growth
• Charged tRNA placed at the A site
• Peptide bond synthesis is followed by ribosomal
  movement
• Starvation
• Uncharged tRNA palced at the A site
• Conformational changes in RelA
• Ribosome remains stationary, iddling reaction
  begins

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• ppGpp inhibits transcription of RNA
• in vitro
• inhibits elongation of txn
• RNA pol pauses
• ppGpp inhibits transcritpion of rRNA
• inhibits initiation at rRNA loci (rRNA promoters)

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ppGpp controls initiation of rRNA

• Figure 11.13

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Control at transcription level

• Stringency
• ppGpp is catalyzed by relA
• ppGpp pauses RNApol
• Elongation/initiation inhibited
• Txn decreases

• Normal growth
• charged tRNA in the A site
• RelA is inactivated
• initiating nts present
• txn of ribosomal RNA begins
• Txn increases

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• Regulatory circuits
• Positive and negative regulators
• Small molecule effectors
• Regulators
• Expression
• Transcription
• Translation

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Control at Translational level

• Regulation of translation
• Regulatory circuits may act at translational
  level
• Repressor binds to the ribosome binding site
• Competition for site between the regulator and
  the ribosome

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Repressor and ribosome compete for binding site
Figure 11.14
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Translational repressors
Figure 11.15
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Translational regulation via secondary structures
of RNA transcript Fig 11.16
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Autogenous Regulation of r-operons
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r-protein synthesis controlled by rRNA
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Phage T4 p 32 is controlled by an autogenous
circuit

• P32
• Involved in genetic recombination, DNA repair and
  replication
• Exercises its functions by binding to single
  stranded DNA
• Inactivating mutations cause overproduction of P32

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Aoutogenous circuit of p32
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P32 binding efficiency important for
autoregulation
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P32 Autogenous Regulation

• How is p32 efficiency regulated?
• Binding affinity
• any RNA lt p32mRNA lt ssDNA (100X)

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Autoregulation of macromolecules
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• Regulatory circuits
• Positive and negative regulators
• Small molecule effectors
• Regulators
• Expression
• Transcription
• Translation

Autogenous regulation
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Regulation at transcription level

• Template recognition
• Initiation
• Elongation
• Termination

CRP, lac operon
ppGpp
Trp operon
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(No Transcript)
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Attenuation/Termination

• Attenuation
• Ability of RNA pol to read through intrinsic
  termination signals
• RNA conformation can be altered
• Secondary structures
• RNA clevagae
• (may yield to changes in the secondary structure)
• Allosteric changes of nucleic acids

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Regulation of trp operon in B.subtilis
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TRAP

• TRAP- Tryptophan Activated Protein
• 11 subunits
• Each subunit binds to one tryptophan aa and one
  trinucleotide
• RNA binds around the protein complex forming an
  alternative secondary structure
• TRAP-terminator protein that responds to the
  presence of trp

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Regulation of trp operon in B.subtilis
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Regulation of trp operon

• Uncharged tRNA trp activates Anti-TRAP
• Anti-TRAP binds to TRAP protein
• TRAP can not bind to RNA to create an intrinsic
  termination
• Trp operon
• tRNA trp Activation
• tryptophan Inactivation

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Trp operon regulation in E. coli
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Trp operon in E. coli
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Regualtory regions in trp operon

• Promoter
• Operator
• Repressor protein TrpR binds to repress txn
• Txn is stopped if a terminary structure is formed
  downstream
• Leader
• peptide that contains two succesive trp
• Attenuator
• 5 of structural genes, site of termination
  structure

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Regulation of trp operon

• Attenuation
• decreases txn at an average of 10X.
• Tryptophan is present, only 10 of the RNA pol
  can procees.

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• Tryptophan is absent
• the repressor is released
• the attenuator is not formed
• the expression is increased
• 70X by derepression
• 10X by attenuation
• Total of 700X!!

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Termination controlled by secondary structures
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Ribosome movement affects secondary RNA structue
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Trp operon regulation

• RNA pol pauses at base 90
• Remains paused until the leader peptide is
  translated
• Secondary strucure is determined before the RNA
  pol reaches the attenuation site

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Trp operon regulation
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Regulation of trp operon

• Tryptophan is absent
• The repressor is released
• The attenuator is not formed
• The expression is increased
• 70X by derepression
• 10X by attenuation
• Total of 700X!!

• Tryptophan is present
• RNA pol pauses at base -90
• RNA pol remains paused until the leader peptide
  is translated
• Secondary strucure is determined before the RNA
  pol reaches te atenuation site
• The repressor bind to the operator
• Txn of the operon is inhibited

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• Regulatory circuits
• Positive and negative regulators
• Small molecule effectors
• Regulators
• Expression
• Transcription
• Translation

Autogenous regulation
Trp Operon