Unit 8: gene regulation 26.5-

Question 1

Mutations in the lac operator cause constitutive expression in what genes?

Answer 1

all three lac structural genes (ZYA)

Question 2

FILL IN THE BLANK: if the mutation affects the expression of all the regulated structural genes then it is most likely a ___________ mutation.

Answer 2

if the mutation affects the expression of all the regulated structural genes then it is most likely a CIS-ACTING mutation.

Question 3

FILL IN THE BLANK: Mutations in the Lac I gene eliminate the ____________ product.

Answer 3

TRANS-ACTING; meaning they are trans-acting and affect expression of all lacZYA clusters in the bacterium.

Question 4

What are uninducible mutants?

Answer 4

mutants that cannot be expressed at all are called uninducible

Question 5

Mutations in the lac promoter cause what?

Answer 5

in the case of the Lac operon mutations in the promoter abolish the expression of the Lac ZYA genes and is cis-acting

Question 6

Mutations to the operator gene results in constitutive expression, why is this the case?

Answer 6

because these mutations result in the inability of the repressor protein to bind to the operator. as a result the RNA polymerase is no longer blocked by the repressor protein.

Question 7

What is a cis-dominant?

Answer 7

a site or mutation that affects the properties only of its own molecule of DNA, often indicating that a site does not code for trans-acting diffusible product

Question 8

Mutations that eliminate lac I function cause constitutive and are _____?

Answer 8

recessive (lacI-)

Question 9

Mutations in the DNA-binding site of the repressor are constitutive because _____?

Answer 9

the repressor cannot bind the operator

Question 10

what does it mean when a lac I gene is mutated in the recessive lacI-?

Answer 10

it means that if a normal Lac I plus gene is introduced function can be restored even in the presence of a defective lac I gene.

Question 11

Give an example of dominant negative and explain why it’s considered a dominant negative.

Answer 11

LacI-D mutants abolished the ability to turn off the gene by damaging the site in the operator binding site of the repressor. this is considered a dominant mutation because if you have a mix of mutant and wild type in some units in the tetramer, a single lac I - D mutant subunit can inactivate the entire tetramer even if the other subunits are wild type.

Question 12

what might happen if there are mutations to the inducer binding site?

Answer 12

If there are mutations in the inducer binding site on the repressor, then the inducer cannot bind, which means that the repressor cannot be inactivated. this in effect will prevent the RNA polymerase from binding to the promoter and the genes will not be transcribed this is an example of a uninducibility these mutations are called Lac IS

Question 13

What is interallelic complementation?

Answer 13

the change in the properties of a heteromultimeric protein brought about by the interaction of subunits coded by two different mutant alleles.

from transcripts: If you have two different alleles of the Lac I gene the subunits made by each can come together to form a heterotetramer instead of a homotetramer. the properties of this hetero tetramer can be quite different from that of the homotetramer. this type of interaction is called Interallelic complementation the mixed protein can be more or less active than the Homotetramer

Question 14

What is negative complementation? Give an example of when we see negative complementation

Answer 14

this occurs when interallelic complementation allows a mutant subunit to suppress the activity of a wild-type subunit in a multimeric protein

EXAMPLE: when lacI-d mutations occur in the DNA-binding site.

this is seen in the case of the lack minus D mutant where the mutation is dominant negative because even when paired with the wild type allele resulting tetramer cannot bind DNA normally and so the operon will be expressed

Question 15

What are the units of a single repressor ssubunit?

Answer 15

1) N-terminal DNA-binding domain
2) a hinge
3) the core of the protein

Question 16

Describe the structure of the DNA-binding domain

Answer 16

The DNA binding domain contains 2 alpha helices, separated by a turn. The helical regions bind to the major groove of the DNA through interaction with specific bases. The DNA binding domain is linked to the main core body of the protein by a hinge sequence

Question 17

The lac repressor subunit “core” is made up how many subdomains? What is located in the core?

Answer 17

Core subdomain 1 and core subdomain 2

• The inducer binding site lies between these two cores and subdomains. The C terminus of the core contains an Alpha Helix which is the multimerization domain. tetramers are held together by the C terminal bundle of four helices

Question 18

How do monomers form a dimer? and how do dimers form a tetramer?

Answer 18

DIMER: momoners form a dimer by making contacts between core subdomains 1 and 3. Each dimer has two DNA binding domains at one end and the tetramerization helices at the other end.

TETRAMER: dimers form a tetramer by interactions between the tetramerization (c terminal) helices. the tetramer has four inducer binding sites and two sets of DNA binding sites

Question 19

TRUE OR FALSE: Different types of mutations occur in different domains of the repressor protein.

Answer 19

TRUE

Question 20

Where does the lacI-D mutation lie and what do they cause?

Answer 20

in the DNA binding part of the protein. these mutations prevent mixed tetramer from binding to the operator; fewer number of binding sites would mean a reduced affinity for the operator.

Question 21

Where do the recessive lac I minus mutations occur and what do they cause ?

Answer 21

anywhere in the core of the protein. these mutations inactivate the protein so the protein cannot repress the Lac operon.

Question 22

Where are the lac IS mutations and what do they cause?

Answer 22

these mutations are mapped to the core subdomain 1, extending from the inducer binding site to the hinge

Lac IS mutants cannot bind or cannot respond to the inducer.

Question 23

The lac repressor protein binds to the double-stranded DNA sequence of the operator, how does the lac repressor recognize a specific sequence?

Answer 23

the Lac repressor recognizes the operator which is a palindromic sequence ( an inverted repeat). The sequence consists of two repeats, with each repeat covering half of the operator. the axis of symmetry separates the two inverted repeats. The DNA binding domain of each repressors subunit binds to 1/2 side of the operator, so two DNA binding domains of the dimer will bind the entire operator. AKA: (Each inverted repeat of the operator binds to the DNA-binding site of one repressor subunit

Question 24

How can the specific bases that interact w/ the repressor be identified?

Answer 24

The specific bases that interact with the repressor can be identified by mutating these residues and observing if these mutations alter the binding of the repressor.

there are eight sites at which constitutive mutations occur between +5 and +17. just as in the case of the promoter a small number of essential specific bases within a larger region are responsible for the sequence specific binding of the protein to DNA

Question 25

What conformational changes occurs when the repressor is not bound to the inducer?

Answer 25

the two DNA binding domains of the dimer insert into successive terms of the major groove. This is thought to increase the affinity to the operator. also the hinge Helix is inserted into the minor groove of the operator now this causes the DNA to bend about 45 degrees. This bending of the DNA positions the major group near the Helix-turn-Helix.

Question 26

What conformational changes occurs when the inducer binds to the repressor?

Answer 26

When an inducer binds the core there’s a conformational change that disrupts the hinge and the headpiece is no longer in an orientation which is high affinity to the operator. this will result in the repressor no longer staying bound to the operator

(reduces affinity for DNA and releases it from the operator)

Question 27

It has been shown that the repressor dimer will be sufficient to bind the full length of the operator. so why would the dimers need to come together to form a tetramer?

Answer 27

the additional pair of DNA binding domains of the tetramer bind to another operator site so the tetramer can bind two operators simultaneously.

From slide: each dimer in a repressor tetramer can bind an operator, so that the tetramer can bind two operators simultaneously

Question 28

There are two additional operator sites in the initial region of the Lac operon. What are they?

Answer 28

The first operator site 01 is located just at the start of the lac Z gene and has the strongest affinity to the repressor. the other two operator sites O2 and O3 are weaker sequences with 02 located downstream of the start side lac Z, while 03 is upstream of operator 01 within the lac I gene`

Question 29

What is required for full repression?

Answer 29

it requires the repressor bind to the primary operator at the lacZ promoter as well as an additional operator 02 or 03.

Question 30

How is a DNA loop generated by a repressor?

Answer 30

When the repression tetramer binds to two operator sites that are separated between each other by a distance it generates a DNA loop

Question 31

What occurs to the efficiency of the repressor if 02 or 03 (additional binding sites) are deleted? what if both are deleted?

Answer 31

the efficiency of the repression is reduced two fold or four fold. However if both 02 and 03 are deleted, repression is reduced 50 fold.

Question 32

TRUE OR FALSE: binding of the repressor to the operator stimulate the binding of the RNA polymerase but prevents transcription

Answer 32

True

Question 33

Where do the LacI-d mutations lie? what do these mutations cause?

Answer 33

1) Lac I-d mutations lie in the DNA binding part of the protein.
2) these mutations prevent mixed tetramer from binding to the operator and fewer number of binding sites would mean a reduced affinity for the operator

Question 34

Where does the recessive Lac I- mutation occur? What do these mutations inactivate?

Answer 34

The recessive Lac I- mutations occur anywhere in the core of the protein. These mutations inactivate the protein so the protein cannot represse the lac operon

Question 35

Lac IS mutations cannot respond to what? where do these mutations occur?

Answer 35

Lac IS mutants cannot bind or cannot respond to the inducer. these mutations are mapped to the core subdomain 1, extending from the inducer binding site to the hinge

Question 36

How does the Lac repressor recognize specific sequences?

Answer 36

the manner in which the Lac repressor recognizes a specific sequence is due to a particular feature in the sequence. the operator is a palindromic sequence which is an inverted repeat. The sequence consists of two repeats, with each repeat covering half of the operator. the axis of symmetry separates the two inverted repeats

Question 37

How does the lac repressor bind to the operator?

Answer 37

lac repressor protein binds to the double-stranded DNA sequence of the operator

The DNA binding domain of each repressors subunit binds to 1/2 side of the operator, so two DNA binding domains of the dimer will bind the entire operator. The specific bases that interact with the repressor can be identified by mutating these residues and observing if these mutations alter the binding of the repressor. there are eight sites at which constitutive mutations occur between +5 and +17. just as in the case of the promoter a small number of essential specific bases within a larger region are responsible for the sequence specific binding of the protein to DNA

Question 38

what happens to the repressor when it is not bound to the inducer?

Answer 38

when the repressor is not bound to the inducer the two DNA binding domains of the dimer insert into successive terms of the major groove. This is thought to increase the affinity to the operator. also the hinge Helix is inserted into the minor groove of the operator now this causes the DNA to bend about 45 degrees. This bending of the DNA positions the major groove near the Helix-turn-Helix.

Question 39

What happens when the inducer binds to the core

Answer 39

When an inducer binds the core there’s a conformational change that disrupts the hinge and the headpiece is no longer in an orientation which is high affinity to the operator.this will result in the repressor no longer staying bound to the operator

aka
binding causes a change in repressor conformation that reduces its afinity for DNA and releases it from the operator

Question 40

the repressor dimer is sufficient to bind the full length of the operator. so why would the dimers need to come together to form a tetramer?

Answer 40

the additional pair of DNA binding domains of the tetramer bind to another operator site so the tetramer can bind two operators simultaneously. there are two additional operator sites in the initial region of the Lac operon. Full repression requires the repressor to bind to an additional operator downstream or upstream as well as the primary operator at the lacZ promoter.

Question 41

Which operator site has the strongest affinity to the repressor? Which two operator sites have weaker affinity?

Answer 41

The first operator site 01 is located just at the start of the lacZ gene and has the strongest affinity to the repressor. The other two operator sites O2 and O3 are weaker sequences with 02 located downstream of the start side lac Z, while 03 is upstream of operator 01 within the lac I gene

Question 42

What does full repression require? What happens if 02 and 03 are deleted?

Answer 42

full repression requires the repressor to bind to the primary operator at the lacZ promoter as well as an additional operator O2 or O3. When the repression tetramer binds to two operator sites that are separated between each other by a distance it generates a DNA loop between the two sites as shown in this figure. binding at the additional operator sites affects the level of repression. if O2 or O3 are deleted the efficiency of the repression is reduced two fold or four fold. However if both 02 and 03 are deleted, repression is reduced 50 fold.

Question 43

Fill in the blank: Binding of the repressor to the operator stimulates the binding of the RNA polymerase but prevents __________.

Answer 43

: Binding of the repressor to the operator stimulates the binding of the RNA polymerase but prevents TRANSCRIPTION

Question 44

TRUE OR FALSE: Proteins that have a high affinity for a specific DNA sequence have a high affinity for other DNA Sequences

Answer 44

FALSE:

Proteins that have a high affinity for specific DNA sequence, have a LOW affinity for other DNA sequences.

Question 45

How does the affinity of the lac repressor to the operator differ from the affinity of the repressor binding to any random site on DNA?

Answer 45

the affinity of lac repressor for the operator is 2x10^13 while the binding to any random DNA side is much less, 2x10^6. Thus, the repressor binds to the operator with 10^7 times higher specificity than a random site.

Question 46

How does induction affect the affinity of the lac repressor to the operator?

Answer 46

induction reduces the affinity for the operator to 10^4 times that of low affinity sites.

-the operator is bound 96% of the time with the repressor effectively repressing the operon although not completely. when the inducer is present only 3% of the operators bound and the operon is induced

Question 47

What does the large number of low-affinity sites ensure?

Answer 47

it ensures that all repressor proteins is bound to DNA

Question 48

How does the lac repressor bind to the operator?

Answer 48

repressor binds to the operator by moving from a low-affinity site rather than by equilibrium solution.

Question 49

How does the repressor behave when the inducer binds to it vs when it is removed?

Answer 49

• When the inducer binds to the repressor it alters its confirmation so it can no longer bind the operator and the inducer plus repressor molecule is thought to bind at random low affinity sites on the DNA.
• when the inducer is removed, the repressor returns to the active form and moves from the random site to the operator by sliding or by direct displacement

Question 50

What is catabolite repression? How is mediated?

Answer 50

catabolite repression is the ability of glucose to prevent the expression of a number of genes.

-This is mediated through the second messenger cyclic AMP (cAMP) and CRP

Question 51

What is CRP?

Answer 51

• Catabolite Repressor Protein (CR)

- it is an activator protein that binds to a target sequence at a promoter

Question 52

What is the first layer of control for the lac operon (is it positive/negative inducible/repressor) and what is the second layer of control for the lac operon?

Answer 52

the Lac operon is negative inducible with transcription being turned on by the inducer allolactose. Now In addition to this control the Lac operon is under a second layer of control. it cannot be turned on by lactose if there is a sufficient supply of glucose. aka catabolite repression

Question 53

Some promoters need an additional protein entity before the RNA polymerase can initiate transcription; these proteins are what type of regulators and why?

Answer 53

these proteins are positive regulators as their presence is required to initiate transcription. CRP is one such protein

Question 54

How is a dimer of CRP activated?

Answer 54

by a single molecule of cAMP

Question 55

How is cAMP levels controlled?

Answer 55

it is controlled by the level of glucose in the cell; a low glucose level allows cAMP to be made. A high glucose levels inhibits cAMP production

Question 56

How does CRP activate RNA polymerase?

Answer 56

CPR interacts w/the C terminal domain of the alpha subunit of RNA polymerase to activate it

Question 57

Fill in the blank: When glucose levels are high, there is ___________ cyclic AMP, and CRP is kept ________ b/c there is not enough _______ to bind to CRP.

Answer 57

When glucose levels are high, there is LOW cyclic AMP, and CRP is kept INACTIVE b/c there is not enough cAMP to bind to CRP.

Question 58

Fill in the blank: Glucose ______ the transcription of operons that require CRP.

Answer 58

Glucose inhibits the transcription of operons that require CRP. it does so by reducing the level of cyclic amp

Question 59

What happens when glucose levels are low in the cell?

Answer 59

when the glucose levels fall the cyclic amp levels increase and they bind a CRP activating it and start transcription of the Lac operon.

Question 60

What are the two ways that CRP can activate transcription?

Answer 60

there are two ways that CRP can activate transcription one it can bind the C terminal domain of the Alpha subunit of the RNA polymerase to activate it or can alter the DNA structure to help the polymerase to bind to it

Question 61

How does the trp operon differ from the lac operon?

Answer 61

1) Unlike the lac operon which is negative inducible, the trp operon is a negative repressible
2) Another difference is that the Lac repressor binds only to the operator of the Lac ZYA gene cluster. The trp repressor on the other hand is an example of repressors that bind to more than one operator and can thus control several dispersed gene clusters.
3) Lac repressor is unregulated and the Trp is autoregulated

Question 62

How is trp repressor activated?

Answer 62

Trp is negatively controlled by the level of its product the amino acid tryptophan. The inactive trp repressor is activated by the amino acid tryptophan

Question 63

what genes does the trp repressor bind?

Answer 63

The trp repressor is a small homodimeric protein that can bind the operator of three unlinked gene clusters.

1) The first cluster is the structural genes of the trp EDCBA which codes for the enzymes needed to synthesize tryptophan.
2) The second gene is the trpR regulated gene, which is a gene that encodes for the trp repressor. The repressor protein acts to reduce its own synthesis.
3) Finally the trp repressor binds to the operator for the third set of genes, the aroH genes, which code for one of the enzymes involved in the synthesis of the aromatic amino acids.

Question 64

How does Trp show autoregulation?

Answer 64

The trpR regulator gene is repressed by its own product, the trp repressor. Thus, the repressor protein acts to reduce its own synthesis: It is autoregulated.

Question 65

what is the role of the trp repressor?

Answer 65

to prevent RNA polymerase from initiating transcription at the promoter

Question 66

what is meant by the trp operon being neg. repressible?

Answer 66

this means that the trp repressor is made as an inactive negative regulator. repression means that the gene product tryptophan is a co-regulator for the trp repressor.

Question 67

What happens when tryptophan levels are up?

Answer 67

when the tryptophan levels are up, two molecules bind to the dimeric trp repressor, alter its conformation to the active form, which can now bind to the operator region.

Question 68

What is the second level of control of the trp operon?

Answer 68

Besides the trp operon being under the control of the gene product on negative repressible it is also under second level of control called attenuation

Question 69

What is attenuation

Answer 69

This means that the operon can be regulated by controlling termination of transcription at a site located before the first structural gene. This site is called attenuator and is in the 5’ leader sequence of the mRNA

Question 70

What happens if transcription terminates at the attenuator site? What if it doesnt terminate at this site?

Answer 70

if transcription terminates at the attenuator site then the structural genes do not get transcribed. however if transcription does not get terminated at the attenuator site then the polymerase continuous transcription of the gene cluster.

Question 71

What does termination at the attenuator depend on?

Answer 71

termination at the attenuator depends on the levels of trp tRNA. If there are high levels of trp tRNA then termination is sufficient. if there are low levels of trp tRNA, termination does not occur and the structural genes get transcribed.

Question 72

How is termination dependent on changes of the secondary structure in the attenuator mRNA?

Answer 72

termination is achieved by changes in the secondary structure of the attenuator mRNA. if the ribosome translates the leader sequence a Terminator hairpin forms and the polymerase terminates transcription before the structural genes get transcribed.

if the ribosome does not translate the leader sequence the hairpin secondary structure does not form and the polymerase can continue transcription of the gene cluster. the mechanism of antitermination depends on the levels of trp-tRNA which influenced the rate of ribosome movement in the leader region

Question 73

What is the attenuator?

Answer 73

intrinsic terminator that is located between the promoter and the first gene of the trp cluster

Question 74

What happens in the absence of Trp-tRNA?

Answer 74

the absence of Trp-tRNA suppresses termination and results in a 10x increase in transcription

Question 75

What happens if level of trp-tRNA are low? What if they are high? What if they are absent?

Answer 75

termination of attenuation depends on the level of trp tRNA. if there is an adequate amount of trp tRNA termination is sufficient as you see in the bottom panel.

if trp tRNA is low then termination by RNA polymerase does not occur at the termination one site and instead the RNA polymerase would continue transcription. the absence of trp tRNA suppresses termination and leads to a tenfold increase in transcription

Question 76

How does the leader region play a role in attenuator termination?

Answer 76

It has a 14 codon or 14 amino acid open reading frame that begins with AUG codons and is followed by a short region that includes two successive trp codons. if the trp tRNA is low, the ribosomes stall when they reach the trp codon. This stalling means a ribosome cannot translate the reader leader region and an alternate secondary structure prevents the hairpin structure from forming so that now the polymerase can continue transcription.

however if trp tRNA is present, the ribosome is able to translate the leader sequence (leader is translated to a leader peptide) and the attenuator is able to form the hairpin which will cause termination prior to the structural genes being transcribed

Question 77

What is the stringent response?

Answer 77

the ability of bacterium to shut down synthesis of ribosomes and tRNA in poor growth medium

Question 78

What triggers the stringent response?

Answer 78

The trigger for the reaction is the entry of uncharged tRNA into the ribosomal A site

Question 79

Poor growth conditions cause bacteria to produce what?

Answer 79

produce small molecular regulators pppGpp or ppGpp (guanosine tetraphosphate and guanosine pentaphosphate)

Question 80

What happens to the A site when there’s a shortage of amino acids?

Answer 80

uncharged T RNA can enter the A site and this blocks translation. It also causes a protein RelA to catalyze the transfer of the pyrophosphate from ATP to GTP or GDP producing a guanosine pentaphosphate or guanosine Tetraphosphate respectively. These molecules control several reactions but in particular transcription. The major effect is to inhibit the synthesis of stable RNA that is ribosomal RNA and transfer RNA. it competes with ATP during the formation of the open complex during transcription initiation and prevents the reaction from occurring

Question 81

What is a relaxed mutant?

Answer 81

in e.coli these do not display the stringent response to starvation of amino acids or other nutritional deprivation

Question 82

What is the stringent factor?

Answer 82

The protein RelA, which is associated w/ribosomes. It synthesizes ppGpp and pppGpp when an uncharged tRNA enters the ribosome

Question 83

How is the r-protein operon controlled? what does this mean?

Answer 83

translation of the r-protein operon is controlled autogenously , which means that the product of the operon binds to a site on its own polycistronic RNA, preventing further translation

Question 84

What happens to the r-operon when ribosomal RNA is available? What about when there is no rRNA available?

Answer 84

rRNA AVAILABLE:When ribosomal RNA is available, the R operons are being transcribed and translated into r-proteins which then associate with the rRNA. translation of the mRNA continues as long as there is free rRNA to bind to.

NO rRNA AVAILABLE:when there is no rRNA available then free r-protein accumulates which then binds to sites on the mRNA and prevents its own translation