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Chapter 26 The Operon

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Chapter 26 The Operon 26.13 The trp Operon Is Also Controlled by Attenuation attenuation The regulation of bacterial operons by controlling termination of ... – PowerPoint PPT presentation

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Title: Chapter 26 The Operon


1
Chapter 26The Operon
2
26.1 Introduction
  • coupled transcription/translation The phenomena
    in bacteria where translation of the mRNA occurs
    simultaneously with its transcription.
  • operon A unit of bacterial gene expression and
    regulation, including structural genes and
    control elements in DNA recognized by regulator
    gene product(s).

3
26.1 Introduction
  • trans-acting A product that can function on any
    copy of its target DNA. This implies that it is a
    diffusible protein or RNA.
  • cis-acting A site that affects the activity
    only of sequences on its own molecule of DNA (or
    RNA) this property usually implies that the site
    does not code for protein.

4
26.1 Introduction
  • regulator gene A gene that codes for a product
    (typically protein) that controls the expression
    of other genes (usually at the level of
    transcription).
  • structural gene A gene that codes for any RNA
    or protein product other than a regulator.

FIGURE 01 A regulator binds a target site on DNA
5
26.1 Introduction
  • In negative regulation, a repressor protein binds
    to an operator to prevent a gene from being
    expressed.
  • In positive regulation, a transcription factor is
    required to bind at the promoter in order to
    enable RNA polymerase to initiate transcription.

FIGURE 03 Transcription factors enable RNA
polymerase to bind to the promoter
FIGURE 02 A repressor stops RNA polymerase from
initiating
6
26.1 Introduction
  • In inducible regulation, the gene is regulated by
    the presence of its substrate (the inducer).
  • In repressible regulation, the gene is regulated
    by the product of its enzyme pathway (the
    corepressor).

7
26.1 Introduction
  • We can combine these in all four combinations
    negative inducible, negative repressible,
    positive inducible, and positive repressible.

FIGURE 04 Induction and repression can be under
positive or negative control
8
26.2 Structural Gene Clusters Are Coordinately
Controlled
  • Genes coding for proteins that function in the
    same pathway may be located adjacent to one
    another and controlled as a single unit that is
    transcribed into a polycistronic mRNA.

FIGURE 05 The lac operon includes cis-acting
regulator elements and protein-coding structural
genes
9
26.3 The lac Operon Is Negative Inducible
  • Transcription of the lacZYA operon is controlled
    by a repressor protein (the lac repressor) that
    binds to an operator that overlaps the promoter
    at the start of the cluster.
  • constitutive expression A state in which a
    gene is expressed continuously.
  • In the absence of ß-galactosides, the lac operon
    is expressed only at a very low (basal) level.

FIGURE 06 The promoter and operator overlap
10
26.3 The lac Operon Is Negative Inducible
  • The repressor protein is a tetramer of identical
    subunits coded by the lacI gene.
  • ß-galactoside sugars, the substrates of the lac
    operon, are its inducer.
  • Addition of specific ß-galactosides induces
    transcription of all three genes of the lac
    operon.
  • The lac mRNA is extremely unstable as a result,
    induction can be rapidly reversed.

11
FIGURE 07 lac expression responds to inducer
12
26.4 lac Repressor Is Controlled by a
Small-Molecule Inducer
  • An inducer functions by converting the repressor
    protein into a form with lower operator affinity.
  • Repressor has two binding sites, one for the
    operator DNA and another for the inducer.
  • gratuitous inducer Inducers that resemble
    authentic inducers of transcription, but are not
    substrates for the induced enzymes.

FIGURE 08 A repressor tetramer binds the
operator to prevent transcription
13
26.4 lac Repressor Is Controlled by a
Small-Molecule Inducer
  • Repressor is inactivated by an allosteric
    interaction in which binding of inducer at its
    site changes the properties of the DNA-binding
    site (allosteric control).
  • The true inducer is allolactose, not the actual
    substrate of ß-galactosidase.

FIGURE 09 Inducer inactivates repressor,
allowing gene expression
14
26.5 cis-Acting Constitutive Mutations Identify
the Operator
  • Mutations in the operator cause constitutive
    expression of all three lac structural genes.
  • These mutations are cis-acting and affect only
    those genes on the contiguous stretch of DNA.
  • Mutations in the promoter prevent expression of
    lacZYA are uninducible and cis-acting.

15
26.5 cis-Acting Constitutive Mutations Identify
the Operator
  • cis-dominant A site or mutation that affects
    the properties only of its own molecule of DNA,
    often indicating that a site does not code for a
    diffusible product.

FIGURE 10 Constitutive operator mutant cannot
bind repressor protein
16
26.6 trans-Acting Mutations Identify the
Regulator Gene
  • Mutations in the lacI gene are trans-acting and
    affect expression of all lacZYA clusters in the
    bacterium.
  • Mutations that eliminate lacI function cause
    constitutive expression and are recessive
    (lacI).
  • Mutations in the DNA-binding
  • site of the repressor are
  • constitutive because the
  • repressor cannot bind the
  • operator.

FIGURE 11 Defective repressor causes
constitutive expression
17
26.6 trans-Acting Mutations Identify the
Regulator Gene
  • Mutations in the inducer-binding site of the
    repressor prevent it from being inactivated and
    cause uninducibility.
  • When mutant and wild-type subunits are present, a
    single lacId mutant subunit can inactivate a
    tetramer whose other subunits are wild-type.
  • It is dominant negative.

18
26.6 trans-Acting Mutations Identify the
Regulator Gene
  • interallelic complementation The change in the
    properties of a heteromultimeric protein brought
    about by the interaction of subunits coded by two
    different mutant alleles.
  • The mixed protein may be more or less active than
    the protein consisting of subunits of only one or
    the other type.

19
26.6 trans-Acting Mutations Identify the
Regulator Gene
  • negative complementation This occurs when
    interallelic complementation allows a mutant
    subunit to suppress the activity of a wild-type
    subunit in a multimeric protein.
  • lacId mutations occur in the DNA-binding site.
    Their effect is explained by the fact that
    repressor activity requires all DNA-binding sites
    in the tetramer to be active.

FIGURE 12 Negative complementation identifies
protein multimer
20
26.7 lac Repressor Is a Tetramer Made of Two
Dimers
  • A single repressor subunit can be divided into
    the N-terminal DNA-binding domain, a hinge, and
    the core of the protein.
  • The DNA-binding domain contains two short
    a-helical regions that bind the major groove of
    DNA.
  • The inducer-binding site and the regions
    responsible for multimerization are located in
    the core.

21
FIGURE 13 Lac repressor monomer has several
domains
Structure from Protein Data Bank 1LBG. M. Lewis,
et al., Science 271 (1996) 1247-1254. Photo
courtesy of Hongli Zhan and Kathleen S. Matthews,
Rice University.
22
26.7 lac Repressor Is a Tetramer Made of Two
Dimers
  • Monomers form a dimer by making contacts between
    core subdomains 1 and 2.
  • Dimers form a tetramer by interactions between
    the tetramerization helices.

FIGURE 15 Repressor is a tetramer of two dimers
23
26.7 lac Repressor Is a Tetramer Made of Two
Dimers
  • Different types of mutations occur in different
    domains of the repressor protein.

FIGURE 16 Mutations identify repressor domains
24
26.8 lac Repressor Binding to the Operator Is
Regulated by an Allosteric Change in Conformation
  • lac repressor protein binds to the
    double-stranded DNA sequence of the operator.
  • The operator is a palindromic sequence of 26 bp.
  • Each inverted repeat of the operator binds to the
    DNA-binding site of one repressor subunit.

FIGURE 17 The lac operator has dyad symmetry
25
26.8 lac Repressor Binding to the Operator Is
Regulated by an Allosteric Change in Conformation
  • Inducer binding causes a change in repressor
    conformation that reduces its affinity for DNA
    and releases it from the operator.

FIGURE 18 Inducer controls repressor conformation
26
26.9 lac Repressor Binds to Three Operators and
Interacts with RNA Polymerase
  • Each dimer in a repressor tetramer can bind an
    operator, so that the tetramer can bind two
    operators simultaneously.
  • Full repression requires the repressor to bind to
    an additional operator downstream or upstream as
    well as to the primary operator at the lacZ
    promoter.
  • Binding of repressor at the operator stimulates
    binding of RNA polymerase at the promoter but
    precludes transcription.

FIGURE 21 Repressor can make a loop in DNA
27
26.10 The Operator Competes with Low-Affinity
Sites to Bind Repressor
  • Proteins that have a high affinity for a specific
    DNA sequence also have a low affinity for other
    DNA sequences.
  • Every base pair in the bacterial genome is the
    start of a low-affinity binding site for
    repressor.

FIGURE 23 Repressor specifically binds operator
DNA
28
26.10 The Operator Competes with Low-Affinity
Sites to Bind Repressor
  • The large number of low-affinity sites ensures
    that all repressor protein is bound to DNA.
  • Repressor binds to the operator by moving from a
    low-affinity site rather than by equilibrating
    from solution.

FIGURE 24 Repression affects the sites at which
repressor is bound on DNA
29
26.10 The Operator Competes with Low-Affinity
Sites to Bind Repressor
  • In the absence of inducer, the operator has an
    affinity for repressor that is 107 times that of
    a low-affinity site.
  • The level of 10 repressor tetramers per cell
    ensures that the operator is bound by repressor
    96 of the time.
  • Induction reduces the affinity for the operator
    to 104 times that of low-affinity sites, so that
    operator is bound only 3 of the time.

30
26.11 The lac Operon Has a Second Layer of
Control Catabolite Repression
  • catabolite repression The ability of glucose to
    prevent the expression of a number of genes.
  • In bacteria this is a positive control system in
    eukaryotes, it is completely different.
  • Catabolite repressor protein (CRP) is an
    activator protein that binds to a target sequence
    at a promoter.

31
FIGURE 25 CRP binds to a consensus sequence.
32
26.11 The lac Operon Has a Second Layer of
Control Catabolite Repression
  • A dimer of CRP is activated by a single molecule
    of cyclic AMP (cAMP).
  • cAMP is controlled by the level of glucose in the
    cell a low glucose level allows cAMP to be made.
  • CRP interacts with the C-terminal domain of the a
    subunit of RNA polymerase to activate it.

FIGURE 27 Glucose reduces CRP activity
33
26.12 The trp Operon Is a Repressible Operon
with Three Transcription Units
  • The trp operon is negatively controlled by the
    level of its product, the amino acid tryptophan
    (autoregulation).
  • The amino acid tryptophan activates an inactive
    repressor encoded by trpR.
  • A repressor (or activator) will act on all loci
    that have a copy of its target operator sequence.

FIGURE 30 CRP-binding sites are close to the
promoter
34
26.13 The trp Operon Is Also Controlled by
Attenuation
  • attenuation The regulation of bacterial operons
    by controlling termination of transcription at a
    site located before the first structural gene.

FIGURE 33 Termination can be controlled via
changes in RNA secondary structure
35
26.13 The trp Operon Is Also Controlled by
Attenuation
  • An attenuator (intrinsic terminator) is located
    between the promoter and the first gene of the
    trp cluster.
  • The absence of Trp-tRNA suppresses termination
    and results in a 10? increase in transcription.

FIGURE 34 An attenuator controls progression of
RNA polymerase into trp genes
36
26.14 Attenuation Can Be Controlled by
Translation
  • The leader region of the trp operon has a
    fourteen-codon open reading frame that includes
    two codons for tryptophan.
  • The structure of RNA at the attenuator depends on
    whether this reading frame is translated.
  • In the presence of Trp-tRNA, the leader is
    translated to a leader peptide, and the
    attenuator is able to form the hairpin that
    causes termination.

37
26.14 Attenuation Can Be Controlled by
Translation
FIGURE 35 The trp operon has a short sequence
coding for a leader peptide
38
26.14 Attenuation Can Be Controlled by
Translation
FIGURE 36 The trp leader region can exist in
alternative base-paired conformations
FIGURE 37 Tryptophan controls ribosome position
39
26.14 Attenuation Can Be Controlled by
Translation
  • In the absence of Trp-tRNA, the ribosome stalls
    at the tryptophan codons and an alternative
    secondary structure prevents formation of the
    hairpin, so that transcription continues.

FIGURE 38 Trp-tRNA controls the E. coli trp
operon directly
40
26.15 Translation Can Be Regulated
  • Translation can be regulated by the 5' UTR of the
    mRNA.
  • Translation may be regulated by the abundance of
    various tRNAs (codon usage).
  • A repressor protein can regulate translation by
    preventing a ribosome from binding to an
    initiation codon.

FIGURE 39 A regulator may block ribosome binding
41
26.15 Translation Can Be Regulated
  • Accessibility of initiation codons in a
    polycistronic mRNA can be controlled by changes
    in the structure of the mRNA that occur as the
    result of translation.

FIGURE 41 Ribosome movement can control
translation
42
26.16 r-Protein Synthesis Is Controlled by
Autoregulation
  • Translation of an r-protein operon can be
    controlled by a product of the operon that binds
    to a site on the polycistronic mRNA.

FIGURE 43 rRNA controls the level of free
r-proteins
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