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BIOL 2320 - Genetics > Regulation of Gene Expression in Bacteria > Flashcards

Regulation of Gene Expression in Bacteria Flashcards

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1
Q

What is most regulation of gene expression in bacteria?

A

transcriptional regulation

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2
Q

What is constitutive transcription?

A

transcription that is continuously occurring

some genes are needed to continuously perform routine tasks - they are constitutively transcribed

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3
Q

When is transcription of genes regulated?

A

when the genes are needed for responses to environmental conditions and not required ALL the time

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4
Q

What two things does transcription regulation control?

A

the initiation and amount of transcription

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5
Q

What is negative control of transcription?

A

when a repressor protein binds to a regulatory DNA sequence

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6
Q

What does negative control of transcription cause?

A

the repressor binding prevents transcription of a gene or gene cluster

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7
Q

Describe positive control of transcription

A

this occurs when an activator protein binds to a regulatory DNA sequence

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8
Q

What does positive control of transcription result in?

A

initiation of transcription of a gene or gene cluster

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9
Q

What function do repressor proteins have?

A

they exert negative control of transcription by binding to regulatory sequences like operators and blocking transcription initiation

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10
Q

What is an operator?

A

a regulatory sequence that occurs on DNA after the promoter region

activated repressors bind to operators

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11
Q

What binds to operators?

A

activated repressor proteins

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12
Q

What are the 2 active sites on repressor proteins?

A

DNA-binding domain

allosteric domain

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13
Q

describe the DNA-binding domain of a repressor protein

A

one of the active sites on a repressor protein that locates and binds to the operator DNA sequence

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14
Q

describe the allosteric domain of a repressor protein

A

one of the active sites

it binds a molecule or protein which causes a conformational change in the DNA-binding domain

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15
Q

How does positive control of transcription occur?

A

when activator proteins bind to regulatory sequences of DNA called activator binding sites

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16
Q

What do activator proteins bind to? AKA What does CAP bind to?

A

activator binding sites (regulatory DNA sequences)

CAP binds to CAP sites when it is already bound to cAMP

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17
Q

What does activator protein binding facilitate?

A

RNA polymerase to bind at promoter and initiate transcription

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18
Q

What domains does an activator protein have?

A

DNA-binding domain

allosteric domain

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19
Q

How many modes of action are there for positive control of transcription?

A

2

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20
Q

Describe the mode of action for positive control of transcription that includes the allosteric binding site of repressor proteins

A

the DNA-binding domain of an activator protein is inactive until

an allosteric effector compound (ligand) binds to the allosteric domain of the repressor protein and induces a conformational change in the DNA-binding domain of the repressor protein which prevents it from binding to the DNA

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21
Q

Describe the alternative mode of positive control of transcription

A

certain activator proteins have a DNA-binding domain that is converted to inactive conformation by the binding of an inhibitor to the allosteric domain

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22
Q

Describe an operon

A

A cluster of genes undergoing coordinated transcriptional regulation by a shared regulatory region

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23
Q

What genomes are operons common in?

A

bacterial genomes

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24
Q

What is the purpose of operons?

A

to allow coordinated expression of genes required for the same biochemical process

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25
Q

What is the function of the lactose (lac) operon in E. coli?

A

it produces two polypeptides required for lactose metabolism

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26
Q

What carbon source do bacteria prefer? Which will they use if this isn’t present?

A

glucose is preferred carbon source

will use lactose if no glucose is present

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27
Q

What does it mean for a system to be inducible?

A

it is not on all of the time, it will be induced

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28
Q

Is the lac operon system on all the time or is it induced?

A

it is induced only when lactose is available

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29
Q

Describe lactose

A

it is a disaccharide consisting of glucose and galactose and joined by Beta-galactoside linkages

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30
Q

What are the 2 components of lactose? what are they linked by?

A

glucose and galactose are linked by beta-galactoside linkage

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31
Q

What kind of medium can bacteria with the lac+ phenotype grow on?

A

media containing lactose as the only sugar

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32
Q

How do bacteria with the lac+ phenotype grow on media with lactose as the only sugar?

A

they produce:

a permease channel

the beta-galactosidase enzyme

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33
Q

What is the purpose of the permease channel?

A

it allows lactose to enter the cell

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34
Q

What gene codes for the permease enzyme channel?

A

lacY

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35
Q

What is the purpose of the beta-galactosidase enzyme?

A

it breaks down the beta-galactoside linkage in the lactose

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36
Q

What gene codes for beta galactosidase?

A

lacZ

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37
Q

what happens to the glucose in lactose?

A

when lactose is broken down, the glucose enters glycolysis

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38
Q

What function does galactose have in lactose when lactose is breaking down?

A

it is processed to produce glucose

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39
Q

What does the breakdown of lactose produce a small amount of? what is its function?

A

A small amount of allolactose

allolactose acts as an inducer

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40
Q

What does allolactose do?

A

act as an inducer

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41
Q

T or F: bacteria with a lac- phenotype are unable to utilize lactose

A

true

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42
Q

What does lac operon consist of?

A

a multipart regulatory region and three structural genes

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43
Q

What are the 3 structural genes of lac operon?

A

lacZ
lacY
lacA

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44
Q

What 3 things does the regulatory region of the lac operon contain?

A

a promoter that binds RNAP

an operator that binds the lac repressor protein

a CAP binding site that binds CAP-cAMP

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45
Q

What does the operator overlap with? What does this cause?

A

the binding location for RNAP

this causes the blocking of RNAP binding cause the repressor protein binds to the regulatory region

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46
Q

Which structural gene of lac operon encodes for beta-galactosidase?

A

lacZ

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47
Q

Which structural gene of lac operon encodes for permease?

A

lacY

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48
Q

Which structural gene of lac operon encodes for transacetylase?

A

lacA

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49
Q

How are the 3 structural genes transcribed?

A

as a single (polycistronic) mRNA

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50
Q

What does translation of the polycistronic mRNA produce?

A

three distinct polypeptides

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51
Q

T or F: lacA, lacZ, lacY are transcribed individually

A

False! they are transcribed as a single polycistronic mRNA

52
Q

T or F: the lac operon consists of lacZ, lacA, lacY, and lacI

A

false! lacI is not part of the lac operon

53
Q

What is the function of lacI? where is it located in relation to the lac operon?

A

lacI codes for the lac repressor protein and is constitutively expressed

it is located next to the lac operon

54
Q

Is the expression of the lacI gene constitutive or inducible?

A

constitutive

55
Q

T or F: the lac operon is transcriptionally active when there is no lactose available or when glucose is available

A

false!! it is transcriptionally silent

56
Q

Why is the lac operon transcriptionally silent when there’s no lactose available or if glucose is available?

A

if no beta-galactosidase is produced by lacZ, there is no allolactose and the lac repressor protein can bind to lacO and prevent transcription

57
Q

What is an example of negative control of transcription?

A

the lac operon being transcriptionally silent in the absence of lactose or presence of glucose

58
Q

When is transcription of the lac operon induced?

A

when lactose is available to the cell and glucose is not

59
Q

Describe how transcription of the lac operon is induced

A

when lactose is available, beta-galactosidase is produced which degrades the lactose into glucose, galactose, and allolactose

When allolactose is produced, it binds to the lac repressor which forms the inducer-repressor complex and causes a conformational change in the repressor protein that prevents it from binding to the operator

60
Q

What forms when allolactose binds to the lac repressor? What does this do?

A

the inducer-repressor complex forms and causes a conformational change in the DNA-binding domain of the repressor and prevents it from binding to the operator

this allows transcription of the lac operon

61
Q

When does basal transcription occur?

A

When both glucose and lactose are present

62
Q

How much polycistronic mRNAs are transcribed when basal transcription occurs?

A

a small number

63
Q

T or F: basal transcription is sufficient to generate enough copies of the lac operon mRNA for metabolism of lactose

A

false! basal transcription only produces a small number of polycistronic mRNAs

64
Q

Where does positive control of the lac operon occur?

A

at the CAP binding region of the lac promoter

65
Q

What are the two domains of the CAP activator protein?

A

DNA-binding domain that binds to the CAP binding region of the promoter

allosteric domain that binds cAMP

66
Q

What does the DNA-binding domain of the CAP activator do?

A

bind to the CAP binding region of the lac promoter

67
Q

What does the allosteric domain of the CAP activator protein do?

A

binds cAMP

68
Q

What restrictions are placed on CAP binding to the CAP binding region of the lac promoter?

A

cAMP must be bound to the allosteric domain

69
Q

What function does DNA-bound CAP have?

A

it interacts with RNAP and increases its ability to bind to the lac promoter

70
Q

How does glucose prevent activation of lac operon?

A

by modulating levels of cAMP

71
Q

What does cAMP stand for?

A

cyclic adenosine monophosphate

72
Q

How is cAMP synthesized?

A

from ATP by adenylate cyclase

73
Q

T or F: during glycolysis, adenylate cyclase is limited in quantity

A

true

74
Q

What is a result of adenylate cyclase being limited during glycolysis?

A

almost no cAMP is produced = almost no CAP-cAMP can form = inefficient lac gene transcription

75
Q

How does the production of cAMP affect lac gene transcription?

A

cAMP production is limited by the small quantity of adenylate cyclase during glycolysis (which makes cAMP from ATP)

cAMP binds to CAP to allow it to bind to the lac promoter region

if there’s not much cAMP to bind to CAP, the lac gene transcription is not very efficient

76
Q

What happens to cAMP levels when glucose is absent?

A

they are high

77
Q

How does the absence of glucose affect transcription of the lac gene?

A

no glucose = high cAMP

CAP-cAMP complex forms efficiently and can bind to the CAP binding domain

CAP-cAMP binds to the lac promoter to interact with RNAP and increase transcription

78
Q

List the steps of lac gene transcription when glucose is absent

A
  1. cAMP levels rise because adenylyl cyclase is more available
  2. CAP-cAMP complex forms and binds to the CAP site of the lac promoter
  3. allolactose forms as B galatosidase breaks down lactose
  4. repressor protein binds allolactose = conformational change = released from operator
  5. RNAP can bind and transcribe
79
Q

When does no lac operon transcription occur?

A

when glucose is present and there’s no lactose

or

when neither glucose or lactose are present

80
Q

T or F: the lac repressor protein binds irreversibly to the operator sequences and cannot be released from lacO

A

false! it binds reversibly and can occasionally be released

81
Q

How were each of the structural genes of the lac operon discovered?

A

by genetic analysis of the lac operon mutants (Jacob, Monod, Lwoff, et al.)

82
Q

Aside from identifying each of the structural genes and the regulatory region of the of the lac operon, what else did the work of Jacob, Monod, Lwoff, etc. do?

A

created a foundation for the description of transcriptional regulation at the DNA sequence level

83
Q

What are two mutations of the lacI gene?

A

lacI-

lacI^S

84
Q

What is the result of the lacI- mutation?

A

the repressor protein product is unable to bind to the operator

85
Q

What is the result of the lacI^S mutation?

A

the repressor protein is a ‘super-repressor’ and is unable to bind the inducer (allolactose) which means it blocks all transcription

86
Q

What is a mutation of the lacZ gene?

A

lacZ-

87
Q

What is the result of the lacZ- mutation?

A

no functional beta-galactosidase is produced = lactose cannot be broken down

88
Q

What mutations does the lacY gene have?

A

lacY-

89
Q

What is the result of the lacY- mutation?

A

no functional permease protein is produced = lactose cannot be transported across the membrane

90
Q

What mutations does the lacA gene have?

A

no transacetylase is produced

91
Q

What mutations can the lacO gene have?

A

lacO^C

92
Q

What is the result of the lacO^C mutation?

A

the repressor protein cannot bind to the operator = continuous transcription

93
Q

What mutations can the lacP gene have?

A

lacP-

94
Q

What is the result of the lacP- mutation?

A

RNAP cannot be bound or it is bound weakly to the promoter

95
Q

How were lac- mutants synthesized?

A

by treating E. coli with mutagens and running complementation tests

96
Q

What two complementation groups did complementation analysis classify lac- mutations into?

A

bacteria with mutations in the lacZ gene

bacteria with mutations in the lacY gene

97
Q

What organisms was complementation analysis conducted in?

A

partial diploids produced by conjugation between F’ (lac) and F- bacteria

98
Q

How can the genotypes of a partial diploid bacteria be written?

A

I+ P+ O+ Z+ Y- / I+ P+ O+ Z- Y+

99
Q

What is one copy of the operon of a partial diploid unable to produce? What about the other?

A

one copy is unable to produce a functional permease (lacY-)

the other is unable to produce functional beta-galactosidase (lacZ-)

100
Q

How do partial diploids have complementation even with mutations?

A

in combination, the mutations of each copy of the operon complement because the WT allele of each gene is dominant to the mutant

101
Q

What phenotype would I+P+O+Z+Y+ produce?

A

wild type (lac+)

102
Q

What phenotype would I+P+O+Z-Y+ produce?

A

no functional beta-galactosidase = lac-

103
Q

What phenotype would I+P+O+Z+Y- produce?

A

No permease = lac-

104
Q

What phenotype would I+P+O+Z+Y-/ I+P+O+Z-Y+ produce?

A

wild type (lac+)

105
Q

Where constitutive mutants located? Why?

A

to the lacO and lacI regions

because these mutations affect the production of the lacI regulatory protein or its DNA-binding site (lacO)

106
Q

Are lac operator mutations exclusively cis- or trans-acting? what does this mean?

A

cis-acting

means they influence transcription of genes on the SAME chromosome

107
Q

How do lacO^C mutants affect downstream structural genes?

A

OC mutants have altered operator sequences which repressor proteins cannot bind to = structural genes downstream are constitutively expressed because there’s no negative control of transcription

108
Q

In experiments with partial diploids, what resulted from an OC mutation being adjacent to Z+ and an O+ next to a Z-?

A

constitutive expression of Z+

109
Q

In experiments with partial diploids, what resulted from an OC mutation being adjacent to Z- and an O+ next to a Z+?

A

normal expression of Z+

110
Q

In experiments with haploid cells, what resulted from a I- P+ O+ Z+ Y+ genotype?

A

the Z and Y genes are constitutively expressed

111
Q

In experiments with partial diploids, what resulted from I+ P+ O+ Z- Y+ / I- P+ O+ Z+ Y-

A

normal expression of both Z and Y genes

112
Q

Is the regulatory protein produced by lac+ cis- or trans-acting? what does this mean?

A

trans-acting which means it influences transcription of genes on different chromosomes

113
Q

How is the lac+ produced regulatory protein trans-acting?

A

it is able to interact with both operators in a partial diploid (both chromosomes) because it can diffuse through the cytoplasm

114
Q

Describe cis-acting

A

influence on transcription of genes on the same chromosome

115
Q

Describe trans-acting

A

influence on transcription of genes on different chromosomes

116
Q

How can a lacI- gene lead to constitutive transcription?

A

lacI- produces a mutant form of the repressor protein that cannot bind to the operator = transcription cannot be repressed

117
Q

In partial diploids, what happens if both lacI- and lacI+ are present?

A

the gene will function normally and the repressor protein will function normally

118
Q

Is lacI- dominant or recessive to lacI+?

A

lacI- = recessive

lacI+ = dominant

119
Q

Describe the lacI^S mutation

A

results in a non-inducible operon where the Z and Y genes are not expressed

alters the allosteric domain so that allolactose cannot bind to it

120
Q

is the lacI^S mutation dominant or recessive to the lacI+ allele?

A

IS = dominant

I+ = recessive

121
Q

How does mutations of lacP affect transcription of lacZ and lacY?

A

most mutations of lacP reduce or eliminate the transcription of Z and Y

122
Q

What phenotype would result from I-P+O+Z+Y+?

A

the lacI- mutation will cause constitutive transcription

123
Q

What phenotype would result from I+ P+ OC Z+ Y+?

A

constitutive transcription because of the mutant lacOC

124
Q

What phenotype would result from IS P+ O+ Z+ Y+?

A

IS mutation makes transcription not inducible

125
Q

What phenotype would result from I+P-O+Z+Y+?

A

lacP- mutation prevents transcription

126
Q

What is a corepressor?

A

a molecule that binds to the allosteric site of a repressor protein and helps it do its job of repressing gene transcription

127
Q

What is an inducer?

A

a molecule that binds to the allosteric site of a protein and stimulates gene expression

ex. inducer binds to the activator protein to stimulate gene expression
ex. inducer binds to the allosteric site of repressor protein and causes conformational change in DNA binding site which prevents repression of transcription

BIOL 2320 - Genetics (12 decks)

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