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Genes and Bioinformatics > Week 2B - Regulation of Bacterial Gene Expression - The Operon Part II > Flashcards

Week 2B - Regulation of Bacterial Gene Expression - The Operon Part II Flashcards

1
Q

The default of the lac operon is

A

off

• probably not in a lactose environment

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2
Q

lacI

A

always on
• codes repressor
• product binds operator that overlaps where RNA polymerase binds (promoter)
• repressor affected by (allolactose) lactose

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3
Q

The lactose pathway in E. coli operates by

A

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

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4
Q

The lac operon has a second layer of control

A
  • E. coli uses glucose in preference to lactose

* catabolite repression

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5
Q

In the presence of glucose and lactose

A

there is no need for the bacterium to turn on the lac operon

• in the interests of efficiency

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6
Q

Catabolite repression

A

the transcription-level inhibition of the lac operon and a variety of other inducible enzymes by glucose (or more readily used carbon sources)
• the ability of glucose to prevent the expression of a number of genes

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7
Q

Why is the lac operon not switches on in the presence of both glucose and lactose?

A

there is another control that blocks the lac operon from synthesizing

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8
Q

Catabolite repression of the lac operon

A 
gene repressed (activator is inactivated)
inactive activator
-->
add inducer (cAMP)
-->
 active activator (CRP)
-->
induced
(helps RNA polymerase bind - recruited simultaneously)

• in mixed carbon nutrient situations
• lac operon is off
• still bind lactose, no repressor bound but still off = need something else to activate
NEED ACTIVATOR AND REPRESSOR

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9
Q

Catabolite repression is exerted through

A

• a second messenger called cyclic AMP (cAMP)
and
• the positive regulator protein called the catabolite repressor protein (CRP)

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10
Q

cAMP-CRP is

A

an activator that binds to a target sequence at the promoter

• positive-inducible

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11
Q

cAMP is synthesized by the enzyme

A

adenylate cyclase
• using ATP
ATP –> 3’, 5’ - cyclic AMP

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12
Q

Adenylate cyclase

A

synthesizes cAMP

• introduces an internal 3’-5’ phosphodiester bond

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13
Q

Adenylate cyclase activity is repressed by

A

high levels of glucose

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14
Q

High glucose =

A

low cAMP
= low CRP

• indirect way of measuring the amount of glucose

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15
Q

A dimer of CRP is activated by

A

a single molecule of cyclic AMP (cAMP)

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16
Q

cAMP is controlled by

A

the level of glucose in the cell
• low glucose allows cAMP to be made
–> level of cAMP is inversely related to the level of glucose

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17
Q

The level of cAMP is

A

inversely related to the level of glucose

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18
Q

cAMP-CRP interacts with

A

the C-terminal domain of the alpha subunit of RNA polymerase to activate it

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19
Q

RNA polymerase is activated (recruited) by

in catabolite repression of lac operon

A

cAMP-CRP

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20
Q

The cAMP-CRP complex binds to

A

an activator site upstream from the lac promoter

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21
Q

CRP is

A

a dimer of 2 identical subunits

• a CRP monomer contains a DNA-binding region and a transcription-activating region

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22
Q

A CRP monomer contains

A
  • a DNA-binding region

* a transcription-activating region

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23
Q

cAMP-CRP complex binds as

A

a dimer to an activator site
• 61bp upstream of the lacZYA transcriptional start site
• the activator site does not overlap the promoter

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24
Q

cAMP-CRP complex binds as a dimer to

A

an activator site 61bp upstream from the lacZYA transcriptional start site

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25
Q

The activator site where cAMP-CRP binds

A

does not overlap the promoter
(activator sits adjacent to RNA)
• binds before start of promoter = RNA polymerase can bind also

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26
Q

cAMP-CRP induces

A

a large bend when it binds DNA (>90)

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27
Q

What do we need for the lac genes to be expressed

A
  1. allolactose (isomer of lactose) needs to bind to the lac repressor protein so that the repressor cannot bind to the operator
  2. CRP needs to be bound by cAMP (whose presence indicates low glucose) - then CRP can bind to the operator. w/o activated CRP, the lac genes will not be expressed
  3. together these conditions indicate the presence of lactose and the absence of glucose
    - -> the lac operon can be under poth positive (cAMP-CRP) and negative (lac repressor) control
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28
Q

The business of catabolite repression—purpose

A
  • if there’s glucose, catabolite repression blocks the lac operon (wasting energy when can consume glucose instead of lactose)
  • if the glucose is low, cAMP is made –> binds/activates CRP –> recruits RNA polymerase to express the lac operon
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29
Q

The lac operon can be

A 
positively controlled (cAMP-CRP) 
negatively controlled (lac repressor)
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30
Q

Alternative use of energy sources in bacteria

A

carbohydrates

eg glucose vs lactose

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31
Q

Bacteria can also produce

A

amino acids

• if these aren’t present in the medium

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32
Q

Example of synthesis of amino acid by E. coli

A

tryptophan - the trp operon

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33
Q

The trp operon has

A
  • an operator
  • a leader region
  • an attenuator
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34
Q

The trp operon is regulated at the levels of

A
  • transcription initiation
  • elongation
  • termination
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35
Q

The trp system is turned off when

A

tryptophan is added to the E. coli culture

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36
Q

The trp operon is under … control

A

negative repressible

the trp repressor is made as an inactive negative regulator

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37
Q

The trp repressor is made as

A

an inactive negative regulator

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38
Q

The trp repressor requires

A

tryptophan to bind the Trp operator

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39
Q

The trp operon is … controlled

A

negatively controlled by the level of its product
(the amino acid tryptophan)
= autoregulation

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40
Q

Autoregulation

A

the trp operon is negatively controlled by the level of its product - tryptophan

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41
Q

The amino acid tryptophan activates

A

an inactive repressor encoded by trpR

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42
Q

The inactive repressor of the trp operon is encoded by

A

trpR

• repressor = trp1

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43
Q

Coarse on-off switch that turns the tryptophan operon off when

A

tryptophan levels are high

44
Q

Tryptophan…

A

activates the inactive aporepressor
–>
tryptophan-aporepressor complex binds to the operator and represses transcription

45
Q

Tryptophan activates

A 
the repressor
(tryptophan = corepressor)
46
Q

The tryptophan operon’s goal is to

A

synthesize tryptophan

47
Q

Can absorb trp from the environment, but if there’s none in the environment

A

this metabolic pathway (trp operon) converts chorismate to tryptophan

48
Q

By default, the trp operon is

A

on

• off when trp is in the environment

49
Q

In bacteria (prokaryotes), mRNA is

A

transcribed, translated, and degraded simultaneously
1. transcription begins
(5’ end is triphosphate)
2. ribosome begins translation
(on mRNA that’s still attached/being transcribed)
3. degradation begins at 5’ end
(translation still occurring)
4. RNA polymerase terminates at 3’ end
5. degradation continues, ribosome completes translation

50
Q

Expression of mRNA in animal cells (eukaryotes) requires

A

transcription, modification, processing, nucleocytoplasmic transport, and translation
1. transcription starts
5’ end is modified (triphosphate)
2. 3’ end of mRNA is released by cleavage
3. 3’ end is polyadenylated (add A)
4. mRNA is transported to cytoplasm
5. ribosomes translate mRNA

51
Q

The repressor of the tryptophan operon is

A

trp1

52
Q

The repressor is synthesized

A

in an inactive form

on by default, off in trp-rich environment bc trp activates the repressor

53
Q

In a trp-rich environment

A

the operon is switched off
• trp activates repressor
–> sensing mechanism
so switched off by end product

54
Q

The trp operon is switched off by

A

the end product (tryptophan - activates repressor)

• feedback inhibition in a metabolic pathway

55
Q

Tryptophan is sensed at the level of

A

translation

• trp = amino acid used by the ribosome to make a protein

56
Q

In prokaryotes, transcription and translation

A

occur at the same time

57
Q

Bacteria can use translation to

A

control transcription

58
Q

The trp operon is also controlled by

A

attenuation

59
Q

Attenuation

A 
the regulation of bacterial operons by controlling termination of transcription at a site located before the first structural gene
eg of control region
1. promoter (binds RNA polymerase)
2. operator (binds regulator)
3. leader
4. attenuator
60
Q

Example of control region in trp operon / attenuation

A
  1. promoter (binds RNA polymerase)
  2. operator (binds regulator - activator/repressor)
  3. leader
  4. attenuator
61
Q

Attenuation is a

A

second level of control

62
Q

The attenuator

A

a region in the 5’ leader of the mRNA

• contains a small ORF

63
Q

Attenuation in the trp operon means that

A

transcription termination is controlled by the rate of translation of the attenuator ORF

64
Q

Translation + termination

A

only leader is transcribed
• the leader includes the promoter
• the coding region has 2 terminators (attenuation = RNA pol falls off)
so RNA polymerase binds at the promoter and transcribes (the leader) up to the first terminator (attenuation)
• there is a termination hairpin that makes the ribosome fall off

65
Q

No translation + no termination

A

coding region is transcribed
• RNA polymerase goes through the promoter ad terminator 1, stationary ribosome changes secondary structure of mRNA, RNA polymerase falls off at terminator 2

66
Q

An attenuator is located

A

between the promoter and the first gene of the trp cluster

67
Q

An attenuator is

A

an intrinsic terminator

68
Q

The absence of Trp-tRNA

A

suppresses termination and results in a 10x increase in transcription

69
Q

High levels of Trp-tRNA

A

will attenuate or terminate transcription before the structural genes
ie tryptophan is not needed

70
Q

2 tryptophans in the leader peptide, hence

A

trp-tRNAs are needed for translation

71
Q

When Trp-tRNA is not present

A

ribosome stalls at this position

72
Q

The terminator hairpin of the trp operon

A

G-C rich hairpin
/ U-rich single strand
(hairpin followed by U region)

73
Q

Trp-tRNA

A

helps termination transciption
(before structural genes)
• translates tryptophan - which helps repress transcription = control at level of translation

74
Q

Attenuation can be controlled by

A

translation

75
Q

The leader region of the trp operon has

A

a 14-codon orf that includes 2 codons for tryptophan

76
Q

The leader region of the trp operon has a 14-codon open reading frame that includes

A

2 codons for tryptophan

77
Q

The structure of RNA at the attenuator depends on

A

whether this reading frame is translated

ie forms a hairpin or not

78
Q

In the presence of Trp-tRNA

A

the leader is translated into a leader peptide

–> the attenuator is able to form the hairpin that causes termination

79
Q

The attenuator causes termination by

A

forming a hairpin

80
Q

When Trp-tRNA is present

A

ribosome stalls at this position
hairpin is not formed
RNA polymerase transcribes the coding region
–> antitermination is dependent on Trp-tRNA

81
Q

Antitermination is dependent on

A

Trp-tRNA

82
Q

Transciption of leader region

A

DNA: promoter, pause, attenuator, trpE

• polymerase initiates transcription then pauses

83
Q

Tryptophan absent

A

transcription continues into operon
• polymerase elongates
• translation initiates

84
Q

Tryptophan present

A

transcription terminates at attenuator
• termination hairpin forms
• polymerase terminates

85
Q

Attenuation can be controlled by

A

translation

86
Q

The trp leader region

A

can exist in alternative base pair conformations
• 3 hairpin conformations of 4 regions
a) 1 complementary to 2 complementary to 3 complementary to 4 –> 3 loops (bottom top bottom)
b)* 1 pairs with 2, 3 pairs with region 4 –> 2 loops with square between (regions 3 and 4 form the termination hairpin) (2 loops at bottom, square between)
c) region 2 pairs with region 3, leaving regions 1 and 4 unpaired (termination region is single stranded) (1 loop at top, 2 square regions at bottom)
–> leader mRNA can form 3 kinds of loops

87
Q

Leader mRNA can

A

make 3 kinds of loops

–> how far transcribed and tells whether to continue transcription or not

88
Q

The position of the ribosome

A

can determine which structure is formed in such a way that termination is attenuated only in the absence of tryptophan

89
Q

The position of the ribosome can determine which structure is formed in such a way that

A

termination is attenuated only in the absence of tryptophan

90
Q

Tryptophan absent

A

ribosome halts at Trp codons (loop right after)

91
Q

The crucial feature of the ribosome going along is

A

the position of the Trp codons in the leader peptide

• depends on whether regions 3 and 4 can pair to form the terminator hairpin

92
Q

Abundant Trp-tRNA

A
  • ribosomes synthesize leader peptide
  • ribosome continues to UGA codon between regions 1 and 2
  • ribosome now extends over region 2 and therefore prevents it from base pairing
  • results in region 3 pairing with region 4 to form the terminatior hairpin
93
Q

Have Trp-tRNA

tryptophan is abundant

A

the ribosome skips over the trp codon and forms the loop

the loop terminates translation –> no more tryptophan made

94
Q

Low Trp-tRNA

A
  • ribosome stalls at Trp codons (region 1)
  • region 1 is sequestered by the ribosome and can’t pair with region 2
  • region 2 and 3 base pair before region 4 is transcribed
  • no terminator hairpin forms, translation continues and tryptophan is made
95
Q

Don’t have Trp-tRNA

little tryptophan

A
  • ribosome stops to translate tryptophan codons
  • the termination hairpin isn’t made
  • transcription and translation continue
96
Q

There are 4 regions in

A

leader RNA that can make a loop

97
Q

The regions that make the termination hairpin are

A

regions 3 and 4

98
Q

RNA polymerase decides what to do

A

based on the ribosome

99
Q

In the absence of Trp-tRNA, the ribosome

A

stalls at the tryptophan codons
and an alternative secondary structure prevents the formation of the hairpin
–> transcription continues

100
Q

CRP is an

A

activator

101
Q

CRP is an activator which is only able to bind to target sequences when

A

complexed with cAMP which only happens in conditions of low glucose

102
Q

CRP (activator) is only active

A

in levels of low glucose

103
Q

The tryptophan pathway operates by

A

negative repression
• the corepressor tryptophan (the product) activates the repressor protein so that it binds to the operator and prevents expression of the genes that code the enzymes that synthesize tryptophan

104
Q

The trp operon is also controlled by attenuation

A

translational control

105
Q

Attenuation is an example of

A

translational control

106
Q

The trp operon is controlled by

A
  • negative repression (product is corepressor = repressor active, feedback inhibition)
  • translational control (attenuation)
107
Q

The trp operon is oly transcribed when

A

trp is unavailable for the ribosome

while the trp leader transcript (trpL) is constitutively expressed

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