Week 2B - Regulation of Bacterial Gene Expression - The Operon Part II Flashcards
The default of the lac operon is
off
• probably not in a lactose environment
lacI
always on
• codes repressor
• product binds operator that overlaps where RNA polymerase binds (promoter)
• repressor affected by (allolactose) lactose
The lactose pathway in E. coli operates by
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
The lac operon has a second layer of control
- E. coli uses glucose in preference to lactose
* catabolite repression
In the presence of glucose and lactose
there is no need for the bacterium to turn on the lac operon
• in the interests of efficiency
Catabolite repression
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
Why is the lac operon not switches on in the presence of both glucose and lactose?
there is another control that blocks the lac operon from synthesizing
Catabolite repression of the lac operon
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
Catabolite repression is exerted through
• a second messenger called cyclic AMP (cAMP)
and
• the positive regulator protein called the catabolite repressor protein (CRP)
cAMP-CRP is
an activator that binds to a target sequence at the promoter
• positive-inducible
cAMP is synthesized by the enzyme
adenylate cyclase
• using ATP
ATP –> 3’, 5’ - cyclic AMP
Adenylate cyclase
synthesizes cAMP
• introduces an internal 3’-5’ phosphodiester bond
Adenylate cyclase activity is repressed by
high levels of glucose
High glucose =
low cAMP
= low CRP
• indirect way of measuring the amount of glucose
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
• low glucose allows cAMP to be made
–> level of cAMP is inversely related to the level of glucose
The level of cAMP is
inversely related to the level of glucose
cAMP-CRP interacts with
the C-terminal domain of the alpha subunit of RNA polymerase to activate it
RNA polymerase is activated (recruited) by
in catabolite repression of lac operon
cAMP-CRP
The cAMP-CRP complex binds to
an activator site upstream from the lac promoter
CRP is
a dimer of 2 identical subunits
• a CRP monomer contains a DNA-binding region and a transcription-activating region
A CRP monomer contains
- a DNA-binding region
* a transcription-activating region
cAMP-CRP complex binds as
a dimer to an activator site
• 61bp upstream of the lacZYA transcriptional start site
• the activator site does not overlap the promoter
cAMP-CRP complex binds as a dimer to
an activator site 61bp upstream from the lacZYA transcriptional start site
The activator site where cAMP-CRP binds
does not overlap the promoter
(activator sits adjacent to RNA)
• binds before start of promoter = RNA polymerase can bind also
cAMP-CRP induces
a large bend when it binds DNA (>90)
What do we need for the lac genes to be expressed
- allolactose (isomer of lactose) needs to bind to the lac repressor protein so that the repressor cannot bind to the operator
- 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
- 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
The business of catabolite repression—purpose
- 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
The lac operon can be
positively controlled (cAMP-CRP) negatively controlled (lac repressor)
Alternative use of energy sources in bacteria
carbohydrates
eg glucose vs lactose
Bacteria can also produce
amino acids
• if these aren’t present in the medium
Example of synthesis of amino acid by E. coli
tryptophan - the trp operon
The trp operon has
- an operator
- a leader region
- an attenuator
The trp operon is regulated at the levels of
- transcription initiation
- elongation
- termination
The trp system is turned off when
tryptophan is added to the E. coli culture
The trp operon is under … control
negative repressible
the trp repressor is made as an inactive negative regulator
The trp repressor is made as
an inactive negative regulator
The trp repressor requires
tryptophan to bind the Trp operator
The trp operon is … controlled
negatively controlled by the level of its product
(the amino acid tryptophan)
= autoregulation
Autoregulation
the trp operon is negatively controlled by the level of its product - tryptophan
The amino acid tryptophan activates
an inactive repressor encoded by trpR
The inactive repressor of the trp operon is encoded by
trpR
• repressor = trp1
Coarse on-off switch that turns the tryptophan operon off when
tryptophan levels are high
Tryptophan…
activates the inactive aporepressor
–>
tryptophan-aporepressor complex binds to the operator and represses transcription
Tryptophan activates
the repressor (tryptophan = corepressor)
The tryptophan operon’s goal is to
synthesize tryptophan
Can absorb trp from the environment, but if there’s none in the environment
this metabolic pathway (trp operon) converts chorismate to tryptophan
By default, the trp operon is
on
• off when trp is in the environment
In bacteria (prokaryotes), mRNA is
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
Expression of mRNA in animal cells (eukaryotes) requires
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
The repressor of the tryptophan operon is
trp1
The repressor is synthesized
in an inactive form
on by default, off in trp-rich environment bc trp activates the repressor
In a trp-rich environment
the operon is switched off
• trp activates repressor
–> sensing mechanism
so switched off by end product
The trp operon is switched off by
the end product (tryptophan - activates repressor)
• feedback inhibition in a metabolic pathway
Tryptophan is sensed at the level of
translation
• trp = amino acid used by the ribosome to make a protein
In prokaryotes, transcription and translation
occur at the same time
Bacteria can use translation to
control transcription
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 eg of control region 1. promoter (binds RNA polymerase) 2. operator (binds regulator) 3. leader 4. attenuator
Example of control region in trp operon / attenuation
- promoter (binds RNA polymerase)
- operator (binds regulator - activator/repressor)
- leader
- attenuator
Attenuation is a
second level of control
The attenuator
a region in the 5’ leader of the mRNA
• contains a small ORF
Attenuation in the trp operon means that
transcription termination is controlled by the rate of translation of the attenuator ORF
Translation + termination
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
No translation + no termination
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
An attenuator is located
between the promoter and the first gene of the trp cluster
An attenuator is
an intrinsic terminator
The absence of Trp-tRNA
suppresses termination and results in a 10x increase in transcription
High levels of Trp-tRNA
will attenuate or terminate transcription before the structural genes
ie tryptophan is not needed
2 tryptophans in the leader peptide, hence
trp-tRNAs are needed for translation
When Trp-tRNA is not present
ribosome stalls at this position
The terminator hairpin of the trp operon
G-C rich hairpin
/ U-rich single strand
(hairpin followed by U region)
Trp-tRNA
helps termination transciption
(before structural genes)
• translates tryptophan - which helps repress transcription = control at level of translation
Attenuation can be controlled by
translation
The leader region of the trp operon has
a 14-codon orf that includes 2 codons for tryptophan
The leader region of the trp operon has a 14-codon open reading frame that includes
2 codons for tryptophan
The structure of RNA at the attenuator depends on
whether this reading frame is translated
ie forms a hairpin or not
In the presence of Trp-tRNA
the leader is translated into a leader peptide
–> the attenuator is able to form the hairpin that causes termination
The attenuator causes termination by
forming a hairpin
When Trp-tRNA is present
ribosome stalls at this position
hairpin is not formed
RNA polymerase transcribes the coding region
–> antitermination is dependent on Trp-tRNA
Antitermination is dependent on
Trp-tRNA
Transciption of leader region
DNA: promoter, pause, attenuator, trpE
• polymerase initiates transcription then pauses
Tryptophan absent
transcription continues into operon
• polymerase elongates
• translation initiates
Tryptophan present
transcription terminates at attenuator
• termination hairpin forms
• polymerase terminates
Attenuation can be controlled by
translation
The trp leader region
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
Leader mRNA can
make 3 kinds of loops
–> how far transcribed and tells whether to continue transcription or not
The position of the ribosome
can determine which structure is formed in such a way that termination is attenuated only in the absence of tryptophan
The position of the ribosome can determine which structure is formed in such a way that
termination is attenuated only in the absence of tryptophan
Tryptophan absent
ribosome halts at Trp codons (loop right after)
The crucial feature of the ribosome going along is
the position of the Trp codons in the leader peptide
• depends on whether regions 3 and 4 can pair to form the terminator hairpin
Abundant Trp-tRNA
- 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
Have Trp-tRNA
tryptophan is abundant
the ribosome skips over the trp codon and forms the loop
the loop terminates translation –> no more tryptophan made
Low Trp-tRNA
- 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
Don’t have Trp-tRNA
little tryptophan
- ribosome stops to translate tryptophan codons
- the termination hairpin isn’t made
- transcription and translation continue
There are 4 regions in
leader RNA that can make a loop
The regions that make the termination hairpin are
regions 3 and 4
RNA polymerase decides what to do
based on the ribosome
In the absence of Trp-tRNA, the ribosome
stalls at the tryptophan codons
and an alternative secondary structure prevents the formation of the hairpin
–> transcription continues
CRP is an
activator
CRP is an activator which is only able to bind to target sequences when
complexed with cAMP which only happens in conditions of low glucose
CRP (activator) is only active
in levels of low glucose
The tryptophan pathway operates by
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
The trp operon is also controlled by attenuation
translational control
Attenuation is an example of
translational control
The trp operon is controlled by
- negative repression (product is corepressor = repressor active, feedback inhibition)
- translational control (attenuation)
The trp operon is oly transcribed when
trp is unavailable for the ribosome
while the trp leader transcript (trpL) is constitutively expressed
