Metabolic Regulation

1
Chapter 8

• Metabolic Regulation

2
Gene Expression

• Process of determining which gene to be
  transcribed and translated
• Must regulate this process
• Constitutive expression proteins are present at
  about same level in cell regardless of growth
  conditions
• however enzymes for lactose metabolism made only
  is lactose is present in the medium

3
Major Modes of Regulation

• 1) Control of activity of pre-existing enzymes
  using post-translation modification
• 2) Control amount (presence or absence) of enzyme
  using transcriptional regulation transcription
  and translation
• Activity control is rapid but synthesis control
  is slow (may take a while to turn on or off)

4
Regulation

• Post-translational modification requires changes
  to the protein
• Non-covalent enzymes inhibition must
  occasionally decreases enzyme activity, using
  cellular component that is in a metabolic pathway
• Covalent or non-covalent interactions such as
  feedback inhibition and isoenzymes

5
Feedback Inhibition

• Usually regulates an entire biosynthetic pathway
• has many enzymes and substrates that are
  converted to products
• Final product communicates with an earlier step,
  regulates its own biosynthesis
• Inhibit 1st enzyme in path by final product,
  remove intermediates so no substrate for the next
  enzyme
• When end product that built up is used,
  inhibition is removed and synthesis picks up again

6
Allostery

• Allosteric enzyme has 2 binding sites active
  site and allosteric site
• Effector or inhibitor binds the allosteric site
  causing a reversible change in the active site
• Common in anabolic and catabolic pathways,
  especially branched paths

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Proline and Arginine Synthesis

• Both made from glutamic acid
• If either arginine or proline begins to pile up,
  its pathway can be inhibited while the other path
  is unaffected
• Companies exploit mutants that are unable to shut
  off the path and makes large production of amino
  acid for food supplements

8
Isoenzymes

• Isozymes are different proteins that catalyze
  same reaction but with different regulation
• In biosynthetic pathways with feedback inhibition
• DAHP synthase plays role in aromatic amino acid
  biosynthesis
• 3 versions of the protein - each has own effector
  molecule
• enzyme activity falls to 0 when all 3 proteins
  are inhibited, each product will inhibit the
  enzyme activity partially

9
Covalent Modification of Enzymes

• Usually by attaching or removing a small molecule
  
• Adding groups changes shape of enzyme and
  catalyze site, remove the group and return to
  original shape
• add AMP, ADP, PO4, or CH3 are most common

10
Glutamine Synthetase

• Used in assimilation of NH3
• Modified by adenylation AMP
• 2 levels of control 1) feedback inhibition by 9
  different compounds (concerted all nine
  required to shut off) and 2) covalent
  modification controlled by glutamine and
  ?-ketoglutarate
• GS has 12 subunits, each can have AMP added, need
  all 12 subunits with AMP to be inactive
  partially adenylated partial inactive
• AMP added/removed on PII which is the enzyme that
  regulates the GS protein
• glutamine levels decreases, PII is modified GS
  is deadenylated and increases activity
• glutamine levels increases, GS becomes highly
  adenylated and decreases activity
• when nitrogen levels are low, GS increases in
  activity and vise versa
• catalytic rxn of GS uses ATP

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GS Control
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Protein Processing

• Not all post-translational modification is
  reversible like GS
• May require new proteins to be processed before
  they can become active
• removal of f-Met and signal peptide are types of
  post-translational processing

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Inteins

• Unusual form of processing is to discard portions
  of a protein and fuse the fragments to make an
  active protein
• protein splicing and formation of an intein
  (similar to an intron in RNA)
• Occurs in the making of DNA gyrase in M. leprae
• self-splicing

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DNA Binding Proteins

• Use proteins that bind to DNA to regulate
  transcription by negative and positive control
• Interaction between the protein and the DNA is
  either specific (specific nucleotide sequence) or
  non-specific (binds anywhere along the DNA like
  histones)
• if histones are on the DNA then the RNA pol
  cannot transcribe the gene, remove histone see
  transcription
• most proteins interact with DNA in a specific
  manner

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Binding

• Use inverted repeats as binding site no stem
  loop structure
• Proteins are usually homodimeric with 1 dimer
  binding at each repeat sequence
• recognizes specific molecular contacts associated
  with specific base sequences
• DNA binding proteins have several distinct
  conformations

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Helix-Turn-Helix

• 2 ? helices one being the recognition helix
  that interacts with the DNA and the other being
  the stabilizing helix for the first one with
  hydrophobic interactions
• Between the helices is a turn that is 3 AA long
  with Gly as the first one
• Many repressors have this type of structure

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Zinc Finger

• Regulatory protein in eukaryotes
• Contains a Zn molecule
• ? helix recognizes the DNA in the major groove
• Usually 2 Zn-fingers involved in regulation

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Leucine Zipper

• Leucine is spaced every 7 AA so that when 2 are
  brought together they zip up
• Leucine containing helix does not actually
  interact with the DNA, binds to another protein
  that recognizes the DNA

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Regulation

• 2 outcomes
• block transcription which is a negative control
• can be repression or induction
• activate transcription which is positive
  regulation

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Negative Control

• Most enzyme synthesis is regulated by bacterial
  environment controlled by the presence or
  absence of small molecules
• Repression dont make the product if it is
  present in medium, only expend the energy if
  absent and it is needed, usually in anabolic
  enzymes
• Induction opposite of repression make only
  when substrate is present usually in catabolic
  enzymes

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Repression

• Add arginine to medium
• Growth of bacteria continues but arginine
  synthesis tapers off
• Other proteins continue to be synthesized at the
  same level
• Final product of the pathway represses the
  enzymes of pathway

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Induction

• Opposite of repression made only when substrate
  is present usually involve catabolic enzymes
  (repression is anabolic enzyme)
• See in use of lactose by E coli, need to make
  ?-galactosidase to convert lactose to glucose and
  galactose
• If no lactose then enzyme is not made

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Effectors

• Effectors are either inducers or co-repressor
• Inducer substance that initiates enzyme
  synthesis
• Co-repressor substance that represses enzyme
  synthesis
• Both may not be substrate or products of enzyme
• can use an analog use IPTF to induce ?-gal
  production but cant be metabolized
• actually not lactose but rather allolactose (made
  in cell from lactose)

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Mechanism of Repression

• Affect transcription indirectly by combining with
  specific DNA binding proteins
• Co-repressor (Arg) to repressor protein called
  arg-repressor which is an allosteric protein
• repressor is active and bind to DNA near promoter
  operator region
• Operon is a cluster of genes arranged in a
  linear, consecutive fashion under control of
  single operator

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Mechanism of Induction

• Induction is also controlled by repressor
• Repressor is active in the absence of inducer
  blocking transcription
• When inducer is present it binds to the repressor
  and inactivate it, transcription can proceed
• Inhibition of mRNA synthesis by specific
  repressor under control of co-repressor molecules
  negative control

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Positive Control

• Regulator protein activates binding of RNA pol
  and transcription ensues
• Maltose metabolism in E coli make the enzymes
  to catabolize maltose under control of activator
  protein
• must bind maltose (effector) before binding DNA,
  bind DNA not at operator but at activator binding
  site still part of operon

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Binding of Activator Proteins

• Promoters of positive controlled operons do not
  have very good consensus sequences RNA pol with
  sigma factor cant recognize the promoter
• Activator bound to DNA helps the RNA pol
  recognize the promoter by bending the DNA to
  allow access to promoter

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Additional Mechanisms

• Activator protein may also interact directly with
  the RNA pol usually close to the promoter or
  when far from promoter causes a loop to form
• E coli also have operons with multiple types of
  control very complex

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Operons vs. Regulons

• Maltose utilization genes spread through out
  chromosome each with an activator binding site
  that the maltose-activator protein can bind
  controls more than one operon at a time in a
  collection called a regulon
• Also have regulons under negative control
  arginine biosynthesis is a regulon

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Global Regulatory Mechanisms

• Regulatory mechanisms that respond to
  environmental signals by regulating expression of
  many different genes
• Global control of the lac operon E coli can use
  many sources for C and energy but really prefers
  glucose
• global control is catabolite repression or
  glucose effect repress a variety of catabolic
  enzyme gene expression can use other C sources
  to repress but needs to be better energy sources

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Diauxic Growth Curve

• Grow on the best source of energy first and when
  exhausted, switch to the next best one
• Undergoes a lag phase while the proteins
  responsible for catabolism of the next substrate
  are made

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?-Gal

• ?-gal is inducible but also under the control of
  catabolite repression glucose around no ?-gal
• When glucose is gone, then the catabolite
  repression is removed
• use cAMP and CAP to regulate the process

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Catabolite Repression

• Requires an activator protein positive control
  mechanism
• RNA pol binds the DNA only when catabolite
  activator protein (CAP) has bound first
• CAP only binds DNA when cAMP is attached
• cAMP made from ATP by adenylate cyclase
  inhibited by the presence of glucose and
  stimulates the transport of cAMP out of the cell
• Glucose enters, cAMP levels fall so catabolite
  repression isnt by glucose but by deficiency of
  cAMP

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Why Global?

• As long as glucose is present, catabolite
  repression keeps all other catabolic operons
  repressed
• Regulatory region require 2 things to be
  present
• levels of cAMP must be high enough so CAP can
  bind DNA - control
• lactose must be present so that the lactate
  repressor doesnt block operator negative
  control
• only if both are met are the enzymes for lactose
  synthesis made

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Stringent Response or Control

• Bacteria in nature experience transient but
  significant change in available nutrients
• Shift up or shift down depending on nutrient
  state
• Move from rich to defined medium no rRNA or
  tRNA synthesis almost immediately
• Amino acid synthesis is geared up even though no
  protein synthesis need to make proteins for
  amino acids not in food (on existing ribosomes)
• Eventually start to make rRNA but at level
  indicative of cells reduced activity

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Mechanism of Stringent Control

• Guanosine tetra- and pentaphosphate trigger the
  response
• Both are alarmones that occur during the shift
  down (AA excess to starvation)
• Rel A in the 50S ribosome subunit uses ATP to
  make ppGpp and pppGpp
• Increase in uncharged tRNA in pool eventually
  bind ribosome and get ppGpp and pppGpp which
  inhibits rRNA and tRNA synthesis
• Positive activation of some AA operons and
  variety of catabolic operons
• Process also inhibits DNA synthesis, membrane
  lipid synthesis and cell division

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Other Global Control Networks

• Modulons describe group genes that are
  regulated by same regulatory protein even if in
  other regulons
• Stimulon group of genes that all respond to
  same environmental signal even if function not
  related

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Alternative Sigma Factors

• Genes in global control systems are controlled by
  alternative sigma factors
• changes the amount and activity of factors
• control transcription of synthesis/activity of
  each
• concentration of factor modulation by
  transcriptional control
• Activity can be control by other proteins such as
  anti-sigma factors that temporarily inhibits
  sigma factor in response to environmental changes

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(No Transcript)
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Heat Shock Response

• Sudden increase in temperatures
• 32? degradation is inhibited and more operons
  that contains this specific factor
• normally - 32? is degraded in 1-2 minutes after
  synthesis
• 32? controls the heat shock genes to make heat
  shock proteins expressed during the heat shock
  response
• help cells recover from stress heat, EtOH, UV
  radiation
• Temperature shift to permissive - 32? is
  inactivated by DnaK and hsp are reduced

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3 Major Classes in E.coli

• Hsp70 DnaK prevent aggregation of newly
  synthesized proteins and stabilize unfolded
  proteins, keeps amount of 32? in check in
  unstressed cells
• during stress situation DnaK protein does
  salvage and repair, 32? becomes more abundant,
  heat shock proteins become more abundant
• Hsp60 GroEL and Hsp10 GroES
• both are molecular chaperones to correctly fold
  mis-folded proteins
• Minor class also include proteases to remove
  denatured or irreversible aggregated proteins

42
Cold Shock

• Cold temperature also increases proteins called
  cold shock response proteins
• retarding ribosome function
• helicase, nuclease and ribosome-associated
  proteins directly or indirectly interact with
  DNA, RNA and ribosomes to decrease macromolecular
  synthesis
• increase production of compatible solutes to
  prevent or reduce ice formation
• ice nucleation proteins made when approaching 0
  - help to make a less destructive ice lattice

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Quorum Sensing

• Signal for prokaryotes to respond to the
  presence of organisms of same species
• regulatory pathways that are controlled by
  density of cells quorum sensing
• Mechanism requires sufficient cell numbers of
  given species before eliciting a particular
  biological response such a secreting a toxin
• require enough to make an impact cause disease

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Acyl Homoserine Lactone

• GNB use a special acylated homoserine lactone
  (AHL) secrete to outside and reach a critical
  level only when nearby cells are also secreting
  AHL
• functions as inducer that combines with specific
  activator protein, triggering transcription of
  specific genes

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Luciferase

• 1st seen in terms of regulating bioluminescence
  in bacteria
• Vibrio fischeri makes light as a by-product of
  luciferase
• lux operons under control of activator called
  Lux R
• induced when AHL levels are high enough
• AHL synthesized by protein encoded by lux I gene

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Examples of Quorum Sensing

• Pseudomonas aeruginosa triggers a large number
  of unrelated genes when population density
  becomes sufficiently high help bacteria move
  from growth in liquid medium to a semi-solid
  matrix called a biofilm
• increases pathogenicity and prevents antibiotic
  penetration
• Staphylococcus aureus involves proteins on
  surface that damage cells and interfere with
  immune system
• virulence factors under the control of quorum
  sensing system that responds to a peptide
  produced by the organism
• mechanism is quite complex and involves
  regulatory RNA molecule

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Attenuation

• Does not include regulatory proteins binding to
  DNA
• Occurs after the beginning of transcription but
  before completed reduces the numbers of
  transcripts made
• See in regulations of genes controlling certain
  amino acids in gnb

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Tryptophan Operon

• Contains 5 proteins involved in the process of
  Trp synthesis
• More than 1 type regulation
• 1st enzyme anthranilate synthase (trpD/E) under
  negative control
• sequence in the operon is the leader sequence
  encodes a polypeptide that contains tandem Trp
  codons near terminus and functions as an
  attenuator
• if enough Trp in the cell then the tRNA will be
  charged and the leader sequence is synthesized
  that terminates transcription
• if no Trp, leader sequence is not synthesized and
  rest of genes are transcribed

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Control of Transcription

• Translation can regulate transcription
• In bacteria transcription and translation are
  simultaneous processes
• Transcription of downstream DNA is happening
  while translation starts at 5 end communicate
  together

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Trp Suppression

• Nearly formed mRNA into unique stem-loop that
  causes RNA pol to stop
• stem-loop becomes a transcription pause site that
  followed by U-rich sequence to cause termination
• stem-loop forms between 3 and 4 to prevent
  transcription

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Trp Activation

• If the stem-loop is formed between 2 and 3 then
  the polymerase can continue to make genes into
  mRNA to make proteins to synthesize Trp
• See also in other amino acid synthesis pathways

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Trp Synthesis

• Trp attenuation protein
• When plenty of Trp it binds to this protein which
  binds the leader mRNA and causes transcription
  termination
• If Trp is limited, then it cannot bind the
  protein and transcription ensues

53
Translation-Independent Attenuation Mechanism

• Gram positive bacteria Bacillus uses
  attenuation to regulate amino acid biosynthesis
• Also use in other pathways such as pyrimidine
  synthesis different mechanisms but both E. coli
  and Bacillus access amount of pyrimidine

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• E. coli monitor the rate of transcription but
  not translation
• if pyrimidines are not limiting so transcribe the
  leader to form a stem-loop termination, if in low
  concentration then form a non-terminator
  stem-loop that allows for further transcription
• Different mechanism in Bacillus
• RNA binding protein controls an alternative
  stem-loop, pyrimidine in excess then termination
  when low then it allows the making of pyrimidines

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Signal Transduction

• Must be a way that bacteria can receive signals
  from the environment and transmit to a specific
  target
• Effectors is one type of signal
• Most signals move to sensor that transmits the
  signal signal transduction

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Sensor Kinase/Response Regulator

• 2 component regulatory systems
• 1) specific sensor kinase protein in cell
  membrane
• 2) partner respnse regulator protein
• Kinase phosphorylates compounds,
  autophosphorylates itself on a His residue (His
  kinase)
• PO4 is moved to another protein in the cell
  called the response regulator
• DNA-binding protein that regulates transcription
• Feedback loop involves a phosphatase that removes
  the PO4 group
• usually slower process than response regulator
  phosphorylation

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RNA Regulation

• Less often translation levels are controlled, use
  proteins, sometimes it is a regulatory RNA
• E. coli have a number of small RNAs that bind to
  other RNAs or even to small molecules
• signal recognition particle for excreted proteins

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Anti-Sense RNA

• Small RNA (sRNA) activity binding to mRNA by
  complementary basepairing
• Expression enhanced by conditions that no longer
  favor expression of genes they regulate
• form dsRNAs from the mRNA and then cannot be
  translated
• degraded by specific ribonuclease

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Riboswitches

• Unique form of sRNA
• mRNA can bind small molecules, in biosynthetic
  pathways of enzyme co-factors, few amino acids
  and purine bases
• thiamine riboswitch binds thiamine upstream of a
  coding sequence for enzyme that participates in
  thiamine biosynthesis pathway
• Analogous to feedback inhibition riboswitch can
  assume either of 2 structures as well
• binding of thiamine will prevent ribosome from
  binding to make more thiamine
• Found only in a few bacteria, few plants and
  fungi
• remnants of RNA world