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Genes and Bioinformatics > Week 2A - Regulation of the Bacterial Gene Expression - The Operon Part I > Flashcards

Week 2A - Regulation of the Bacterial Gene Expression - The Operon Part I Flashcards

1
Q

Trans-acting factors

A

generally genes, the product of which can function on any copy of its target DNA
• code for tRNA or rRNA
• ie a protein or RNA molecule that diffuses AWAY from the location of synthesis to act elsewhere
DNA
• a DNA sequence that contains a gene
– this gene codes for a protein (or microRNA or other diffusible molecule) that will be used in the regulation of another target gene.

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2
Q

Cis-acting DNA sequences

A

a site that affects the activity ONLY of sequences on its OWN molecule or DNA (or RNA)
• usually implies that the site does not code for protein
• ie promoters, operators, and terminators
• DNA sequence that functions solely as a DNA sequence, have only LOCAL effects

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3
Q

Structural gene

A

a gene that codes for any RNA or protein product other than a regulator

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4
Q

Regulator gene

A

a gene that codes for a product (typically protein) that controls the expression of other genes (usually at the level of transcription) by binding to particular sites on DNA
(cis-acting sequences)

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5
Q

Operators

A

the sites on DNA are usually located just upstream of the target gene

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6
Q

Regulation in a positive manner

A
  • turns the gene ON

* an activator

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7
Q

Regulation in a negative manner

A
  • turns the gene OFF

* a repressor

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8
Q

In negative regulation…

A 
a trans-acting repressor protein
binds to a 
cis-acting operator 
to prevent a gene from being expressed
• repressor and operator overlap
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9
Q

In negative regulation,

in the absence of a repressor, a gene is

A

ON by default

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10
Q

The most common type of regulation in bacteria

A

negative regulation

with a repressor

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11
Q

In positive regulation…

A

a trans-acting transcription factor activator
is required to bind at the
cis-acting promoter
in order to enable RNA polymerase to initiate transcription
• operator and activator don’t overlap

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12
Q

In positive regulation,
in the absence of the positive regulator
(transcription factor)
a gene is…

A

OFF by default

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13
Q

The most common type of regulation in eukaryotes

A

positive regulation

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14
Q

A gene that encodes an enzyme may be regulated by

A

the concentration of the substrate or product

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15
Q

A bacteria avoids synthesizing the enzyme of a pathway in the absence of the

A

substrate (avoid wasting of energy), but is ready to produce the enzymes if the substrate (inducer) should appear

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16
Q

In inducible regulation, the gene is regulated by

A

the presence of its substrate

the inducer

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17
Q

In repressible regulation, the gene is regulated by

A

the product of its enzyme pathway

the corepressor

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18
Q

4 combinations of regulatory circuits

A
  • negative inducible
  • negative repressible
  • positive inducible
  • positive repressible
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19
Q

Induction

Negative control

A 
negative control = act on repressor
active repressor 
repressed 
by inducer
--> repressor inactivated to get induction
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20
Q

Induction

Positive control

A 
positive control = act on activator
inactive activator
induced by inducer
to be active
--> activator activated to get induction
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21
Q

Repression

Negative control

A 
negative control = act on repressor
inactive repressor
binds corepressor
repressor active
--> repressor activated to get repression
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22
Q

Repression

Positive control

A 
positive control = act on activator
active activator
binds corepressor
to become inactive
--> activator repressed to get repression
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23
Q

Unifying theme: regulatory proteins are

A

trans-acting factors that recognize

cis-acting elements usually upstream of the gene

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24
Q

Operon

A

functioning unit of genomic material containing a cluster of genes under the control of a single regulatory signal or promoter
• eg genes for E. coli metabolism

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25
Q

Genes in an operon are transcribed together into a

A

polycistronic mRNA strand

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26
Q

The result of an operon is that all the genes are

A

either expressed together

or not at all

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27
Q

Genes coding for proteins that function in the same pathway are often located

A

adjacent to one another and controlled as a single unit that’s transcribed into a polycistronic mRNA within an operon

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28
Q

lac operon

A

proteins products of this operon enable bacteria to take up and metabolize β-galactoside sugars
(ie lactose)
• has structural genes (lac Z Y A)

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29
Q

lacZ

A

encodes the enzyme β-galatctosidase which breaks down β-galactoside
(lactose to glucose and galactose)

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30
Q

lacY

A

enables β-galactosidase permease

transports β-galactosides into the cell

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31
Q

lacA

A

encodes β-galactoside transacetylase

transfers acetyl groups from acetyl-coA to β-galactosides

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32
Q

β-galactosidase

A

breaks down β-galactoside

disaccharide –> 2 monosaccharides

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33
Q

β-galactoside sugars are

A

• the substrates of the lac operon
• the inducers
(add = genes transcribed
use up = switch off)

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34
Q

Addition of specific β-galactosides

A

induces transcription of all 3 genes of the lac operon

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35
Q

The lacmRNA is extremely

A

unstable - restricts the amount of protein made

–> induction can be rapidly reversed

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36
Q

Promoter

A

binds RNA polymerase

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37
Q

Operator

A

binds repressor or activator

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38
Q

Transcription of the lacZYA operon is controlled by a

A

repressor protein (the lac repressor)
that binds to an operator
that OVERLAPS the promoter at the start of the cluster

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39
Q

The lac repressor protein is a

A

tetramer of identical subunits coded by the laci gene

40
Q

The lac repressor is encoded by the

A

laci gene
which is an independent transcription unit with its own promoter and terminator
• overlaps with the promoter region where RNA polymerase binds
• laci makes the monomer which forms a tetramer

41
Q

The laci gene

A

is an independent transcription unit

with its own promoter and terminator

42
Q

Constitutive expression

A

a state in which a gene is expressed continuously

• eg problem with laci gene = no lac repressor = constitutively expressed

43
Q

In the absence of β-galactosides

A

the lac operon is expressed only at a very low (basal) level

44
Q

An inducer functions by

A

converting the repressor into a form with lower operator affinity
• the repressor is usually bound, so lac usually off until lactose comes along to change the shape of the repressor

45
Q

Repressor has 2 binding sites

A
  • 1 for the operator DNA

* 1 for the inducer

46
Q

Repressor is inactivated by an

A

allosteric interaction in which binding of inducer at its site changes the properties of the DNA-binding site (allosteric control)
• lactose permeates bacterial cell, the binds lac repressor –> changes shape so it has no affinity for the operator

47
Q

The true inducer is

A

allolactose, not the actual substrate of β-galactosidase

48
Q

Gratuitous inducer

A

inducers that resemble authentic inducers of transcription, but are not substrates for the induced enzymes (eg IPTG)
(here resembles lactose but does the same thing)

49
Q

Cis-acting constitutive mutations identify the

A

operator

50
Q

Mutations in the operator cause

A

constitutive expression of all 3 lac structural genes
(repressor binds at operator)
• these mutations are cis-acting and affect only those genes on the contiguous stretch of DNA

51
Q

Mutations in the promoter that prevent expression of lacZYA are

A

uninducible and cis-acting

52
Q

Cis-dominant

A

a site or mutation that affects the properties only of ts own molecule of DNA, often indicating that a site does not code for a specific product

53
Q

Trans-acting mutations identify the

A

regulator gene

54
Q

Mutations in the laci gene are

A

trans-acting and affect expression of all laczYA clusters in the bacterium

55
Q

Mutations that eliminate laci function cause constitutive expression and are

A

recessive

laci-

56
Q

Mutations in the DNA binding site of the repressor are constitutive because

A

the repressor cannot bind the operator

57
Q

Constitutive expression can come from

A
  • mutations in the operator (where the repressor binds)

* mutations in the laci gene

58
Q

The lac repressor is a

A

tetramer made of 2 dimers

59
Q

A single repressor subunit can be divided into

A
  • the N-terminal DNA-binding domain
  • a hinge
  • the core of the protein
60
Q

The DNA-binding domain of the lac repressor contains

A

2 short α-helical regions that bind the major groove of DNA

helix-turn-helix motif

61
Q

The core of the lac repressor contains

A
  • the inducer-binding site

* the regions responsible for multimerization

62
Q

In the lac repressor, monomers form a dimer by making contacts between

A

core subdomains 1 and 2

63
Q

Dimers form a tetramer by interactions between

A

the tetramerization helices

64
Q

The lac repressor is

A

large compared to DNA

65
Q

lac repressor binding to the operator is regulated by

A

an allosteric change in conformation

66
Q

lac repressor binding to the operator

A

• lac repressor protein binds to the double-stranded DNA sequence of the OPERATOR
(operator is palindromic 26bp)
• each inverted repeat of the operator binds to the DNA-binding site of one repressor subunit
• symmetry of the operator matches the symmetry of the repressor
• inducer binding causes a change in repressor conformation that reduces its affinity for DNA and releases it from the operator
(probably from changing the hinge helices)

67
Q

The operator is

A

a palindromic sequence of 26 bp

• dyad symmetry

68
Q

Each inverted repeat of the operator binds to

A

the DNA-binding site of one repressor subunit

69
Q

Symmetry of the operator matches

A

the symmetry of the repressor

70
Q

Inducer binding causes a change in repressor conformation that

A

reduces its affinity for DNA and releases it from the operator
• probably by changing the hinge helices

71
Q

A dimer of the lac repressor binds

A

2 halves of DNA

• its a tetramer because there’s more than 1 operator to bind to

72
Q

lac operators

A

the original operator (lacO1)
auxiliary operators (weaker)
• 410 bp downstream (lacO2)
• 88 bp upstream (lacO3)

because there’s 3 operators = repressor tetramer binds to more than 1

73
Q

The lac repressor binds to

A

3 operators and

interacts with RNA polymerase

74
Q

Each dimer in a repressor tetramer can

A

bind an operator

• so the tetramer can bind 2 operators simultaneously

75
Q

Full repression requires

A

the repressor to bind to an additional operator
downstream (lacO2) and
upstream (lacO3) as well as
to the primary operator at the lacZ promoter
(bends to bind both the original and lacO3 = locks in transcriptional repression)

76
Q

Binding of the repressor at the operator

A

actually stimulates binding of RNA polymerase at the promoter but precludes transcription

77
Q

The operator competes with

A

low-affinity sites to bind repressor

78
Q

The large number of low-affinity sites ensures

A

that all repressor protein is bound to DNA

at random sites

79
Q

Repressor binds to the operator by

A

moving from a low-affinity site rather than by equilibrating from solution
(previously bound to DNA at random site, moving to operator after its already bound to DNA is easier than coming from solution)

80
Q

In the absence of the inducer

A

the operator has an affinity for repressor that is 10^7 times that of a low-affinity site

81
Q

The level of 10 repressor tetramers per cell ensures that

A

the operator is bound by repressor 96% of the time

82
Q

Induction reduces…

A

the affinity for the operator to 10^4 times that of low-affinity sites, so that the operator is bound only 3% of the time

83
Q

Transcription is regulated by the interaction between

A

trans-acting factors (product of a gene) and

cis-acting sites (DNA sequence)

84
Q

Bacterial genes coding for proteins whose functions are related may be organized in a cluster that is transcribed into a

A

polycistronic mRNA
from a single promoter
• control of this promoter controls all the genes

85
Q

The unit of regulation is called

A

an operon

• contains structural genes and cis-acting elements

86
Q

Initiation of transcription is regulated by interactions that

A

occur in the vicinity of the promoter (at the operator)

87
Q

The ability of RNA polymerase to initiate transcription is prevented

A

by a repressor

88
Q

The ability of RNA polymerase initiate transcription is activated

A

by an activator

89
Q

Genes that are active only when the regulator is bound to them are said to be

A

under positive control

90
Q

Genes that are off when the regulator is bound are said to be

A

under negative control

91
Q

The ability of a repressor to bind its operator sequence is often regulated by

A

small molecules which provide a second level of gene regulation

92
Q

If the repressor regulates genes that encode for enzymes, the system may be

A

induced by the enzyme substrates
(negative inducible - prevents repressor from binding)
–> genes switch ON

93
Q

If the product of the corepressor enables the regulator to bind the operator

A

= negatively repressible

–> gene turned off

94
Q

Binding of the inducer or corepressor to its site on the regulatory protein produces

A

an allosteric change in the structure of the DNA-binding domain of the regulatory protein

95
Q

The lactose pathway in E. coli operates by negative induction

A

• when an inducer, the substrate β-galactoside diminishes the ability of repressor to bind its operator, transcription and translation of the lacZ gene then produce β-galactosidase, the enzyme that metabolizes β-galactosides

Genes and Bioinformatics (10 decks)

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