Microbial Genetics

1
Microbial Genetics

• The Operon
• Regulation of bacterial gene expression

2
Controlling enzymes

• Remember from metabolism lectures (come on, it
  was only 4 lectures back)
• Bacterial cells carry out a huge number of
  chemical reactions catalyzed by enzymes
• Bacterial cells must respond rapidly to changing
  environments e.g., presence of a new carbon
  source
• Enzyme activity can be regulated by feedback
  inhibition of synthesized enzyme
• BUT making enzyme requires energy (1200 ATPs/ave.
  size protein)
• SO it is better to stop synthesizing enzymes that
  are not needed

3
Regulation of gene expression

• Genes through transcription and translation
  direct the synthesis of proteins
• 60-80 of genes are not regulated
• Constitutive fixed rate of expression
• Enzymes that cells need constantly e.g.,
  glycolysis
• Other genes such as those for enzymes involved in
  lactose metabolism or amino acid production are
  regulated and only expressed when required

4
Regulation of gene expression

• The process of transcription plays an important
  role in the ability of bacteria to respond to
  changing environments
• mRNA compared to DNA is inherently unstable, so
  the expression levels of certain proteins can be
  controlled at the level of transcription

5
Transcriptional control

• Two genetic control mechanisms which regulate
  transcription of mRNA and, therefore the
  synthesis of corresponding protein
• Induction-turning on gene expression
• Repression-turning off gene expression

6
Repression

• Inhibition of gene expression
• Often in response to an excess of endproduct,
  shuts down synthesis of the enzyme
• Mediated by regulatory proteins called repressors
• Block RNA polymerase from initiating
  transcription
• Repressible genes are transcribed until they are
  repressed

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Induction

• Turns on transcription of a gene
• A substance which acts to induce transcription of
  a gene is an inducer, often the substrate
• Enzymes synthesized in the presence of inducers
  are called inducible enzymes
• Example lactose utilization genes
• Inducible genes are not transcribed until they
  are induced

8
The operon

• Multiple genes are arranged in the same
  orientation and are closely linked on the DNA
• Genes in an operon are transcribed on a single
  RNA transcript, but are translated individually
  to form multiple proteins
• A mechanism for coordinate control of genes
  involved in a single process by regulating
  transcription of the operon mRNA

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The operon (cont)

• The term operon not only includes the
  structural genes in the operon but also the
  regulatory sequences controlling transcription
• Promoter-site of RNA polymerase binding
• Operator-site of binding of a regulatory protein

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Operons and regulation

• 27 of E. coli genes are in operons
• Many of the genes arranged in operons are
  regulated
• For example,
• genes for the lactose utilization are controlled
  by the presence of lactose
• genes for pathogenesis are often controlled by
  temperature
• Genes for enzymes required all the time are not
  arranged in operons

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Two regulated operons

• Two E. coli operons reflect how operons in
  general are regulated
• The lac operon
• Encodes inducible catalytic enzymes involved in
  the lactose utilization and uptake
• The arg operon
• Encodes repressible anabolic enzymes involved in
  the production of the amino acid arginine

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The lac operon

• Three structural genes
• lacZ ß-galactosidase which splits lactose to
  glucose and galactose
• lacY a permease involved in transport of lactose
  into the cell
• lacA a transacetylase, function unknown
• A regulatory gene lacI
• The LacI protein is a repressor of the lac operon
  
• A promoter where RNA polymerase binds to
  transcribe the operon
• An operator site where the LacI repressor binds
  to block transcription

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Arrangement of the lac operon
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In the absence of lactose

• In the absence of lactose, the repressor binds to
  operator site preventing binding of RNA
  polymerase and transcription

15
In the presence of lactose

• The lac operon is an inducible operon
• Lactose acts as the inducer
• Lactose induces enzyme expression by binding to
  the LacI repressor preventing its binding to the
  operator site

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Catabolite repression

• The glucose effect
• Glucose is more rapidly metabolized than lactose,
  and is a preferred carbon source
• Therefore, the presence of glucose represses the
  lac operon allowing glucose to be used first

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How does glucose control the lac operon?

• Apart from an operator site, the lac operon
  contains a site for binding of CRP (cyclic AMP
  receptor protein). In order for the lac operon to
  be transcribed CRP must be bound.
• Cyclic AMP (cAMP) is a molecule in the cell that
  serves as cellular alarm signal (glucose sensor).
• When available glucose is high, levels of cAMP
  are low. When available glucose is low, levels of
  cAMP are high.

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How does glucose control the lac operon?

• CRP only binds to the lac operon when cAMP levels
  are high (glucose low)
• Therefore, even when lactose is present, if
  glucose is also present, the lactose operon will
  not be transcribed

CRP
RNA polymerase
Glucose present Lactose present
Transcription
No glucose Lactose present
CRP cAMP
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The arg operon

• A repressible operon genes are transcribed until
  turned off or repressed
• Three structural genes argCBH encoding enzymes in
  the arginine biosynthetic pathway

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In the absence of arginine

• The arg operon is transcribed and enzymes for the
  synthesis of arginine are produced

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In the presence of arginine

• Arginine acts as a corepressor and represses its
  own synthesis
• Like feedback inhibition but acts on enzyme
  synthesis rather than activity

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Summary of gene regulation

• The production of many bacterial proteins are
  controlled by regulating transcription
• Functionally related genes are arranged in
  operons to allow coordinate regulation
• Operons can be
• Induced in the presence of substrate
• Repressed in the presence of endproduct

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But wait, theres more!

• Inducible and repressible operons are the major
  ways in which genes are regulated
• Genes can also be regulated by
• Activators regulatory proteins that are
  required for gene transcription
• Attenuators specific sequences in the DNA that
  prevent either transcription or translation of
  the gene except in response to certain conditions