Unit 8: Bacterial Gene Regulation Operons (26.1-26.4)

Question 1

What is coupled transcription/translation?

Answer 1

The phenomena in bacteria where translation of the mRNA occurs simultaneously w/its transcription

Question 2

What is an operon?

Answer 2

a unit of bacterial gene expression and regulation, including structural genes and control elements in DNA recognized by regulator gene product(s)

Question 3

How is transcription controlled/regulated?

Answer 3

by the operon system

Question 4

What two type of sequences does the operon system recognize?

Answer 4

1) one that code for trans-acting products usually proteins

2) and the others are cis-acting DNA sequences

Question 5

What is a trans-acting sequence?

Answer 5

a trans-acting sequence encodes for products usually proteins but could also be RNA molecules that function or affect any copy of its target DNA. This implies that it is a diffusible protein or RNA

Question 6

What is a cis-acting sequence?

Answer 6

A site that affects the activity only of sequences on its own molecule of DNA (or RNA). This property usually implies that the site does not code for protein.

Question 7

What is a regulator gene?

Answer 7

A regulator gene encodes for a product (most of the time a protein) that controls expression of other genes (this control is usually at the level of TRANSCRIPTION and not translation)

Question 8

What is a structural gene?

Answer 8

a structural gene code for any RNA or protein product other than a regulator.

Protein products include structural proteins, enzymes, and regulatory proteins

Question 9

What is meant by negative regulation? what is the role of trans-acting repressor in this form of regulation?

Answer 9

negative regulation means that the repressor protein binds to an operator to prevent a gene from being expressed. (side note: an operator is an example a cis-acting site that is close to the promoter region and is the site for the binding of a repressor protein.)

-in negative control, a trans-acting repressor binds to the cis-acting operator to turn off transcription

Question 10

What is meant by positive regulation? what is the role of trans-acting factor in this form of regulation?

Answer 10

a transcription factor is needed to bind at the promoter in order to enable RNA polymerase to initiate transcription

-in positive control, a trans-acting factor must bind to cis-acting site in order for RNA pol to initiate transcription at the promoter

Question 11

Describe what happens to transcription in the absence and presence of a repressor

Answer 11

when the repressor protein is absent the RNA polymerase binds to the promoter and transcription of the gene commences.

if present, the repressor protein prevents the polymerase from initiating transcription and we say that the gene is turned off.

Question 12

What two regulator systems or systems of control allow bacteria to respond quickly to changing environments? particularly in the supply of available nutrients

Answer 12

1) Inducible regulation - the gene is regulated by the presence of its substrate (the inducer)
2) Repressible regulation - the gene is regulated by the products of its enzymes pathway (the corepressor)

Question 13

What mode of control is being described: The organism has a controlled setup that will permit the production of the enzymes that will breakdown the substrate only if the substrate is present, that is the substrate acts as an inducer to induce expression of the genes needed to act upon that substrate

Answer 13

inducible regulation

Question 14

what is an example of inducible regulation?

Answer 14

An example of this is lactose, where if lactose is present then the genes encoding the enzymes needed for its breakdown will be transcribed or turned on.

Question 15

If enough product is produced, or the product is available from other sources, then the enzymes needed for the synthesis of that product are no longer required and the genes can be turned off - what mode of regulation turns this off?

Answer 15

Repressible regulation

Question 16

In repressible regulation, the product is called what?

Answer 16

corepressor

Question 17

Give an example of repressible regulation

Answer 17

An example of repressible regulation is synthesis of the amino acid tryptophan. if tryptophan is provided to the bacterium in the medium in which it is growing, then the organism does not need to synthesize tryptophan. So the genes that encode for the enzymes that synthesize tryptophan will be turned off

Question 18

Describe negative inducible

Answer 18

a repressor is produced which binds to the operator site preventing transcription.

However in the presence of an inducer, which binds to the repressor, the latter can no longer behave as a repressor and is thus inactivated, allowing for transcription to occur.

Question 19

Describe positive inducible

Answer 19

in positive inducible, the activator is generally inactive until the inducer binds to the activator and activates it, which then binds to a cis-acting site and the RNA polymerase can initiate transcription.

Question 20

Describe negative repressible

Answer 20

Negative repressible, is when the repressor is inactive but in the presence of a corepressor it becomes activated and will prevent transcription of the gene.

Question 21

Describe positive repressible

Answer 21

Positive repressible is when the activator is normally active and is facilitating transcription until a corepressor binds to it, inactivating it and gene transcription is repressed

Question 22

Why would bacteria want to combine positive versus negative control and inducible regulation versus repressible regulation in different combinations?

Answer 22

By using these different combinations a bacterium can have immense control over the different metabolic pathways to allow it to quickly adapt to changing conditions in the environment

Question 23

What do the four combinations (negative inducible, positive inducible, negative repressible, positive repressible) have in common?

Answer 23

In all of this we see trans-acting factors that recognize cis-acting sites, which are usually upstream of the promoter, resulting in activation of gene or repressing it

Question 24

What is one of the key differences between prokaryotes and eukaryotes in the organization of the genome ?

Answer 24

one of the key differences between prokaryotes and eukaryotes in the organization of the genome is that prokaryotes have operons where the structural genes that comprise the operon are under the control of a single regulator. in eukaryotes, generally each gene is individually regulated.

Question 25

when is polycistronic mRNA produced?

Answer 25

when more than one gene will be transcribed off an operon, the transcription unit that the Messenger RNA produces is called a polycistronic mRNA

from slide: genes coding for proteins that function in the same pathway may be located adjacent to one another and controlled as a single unit that is transcribed into a polycistronic mRNA

Question 26

What are the structural genes and the regulatory genes of the Lac Operon? How do these genes differ?

Answer 26

STRUCTURAL GENES:

1) lacZ
2) lacY
3) lacA

REGULATORY GENES:
1) LacI

The Lac I gene is a trans-acting regulatory gene that encodes a repressor and has its own promoter and terminator

Lac Z, Y, and A, on the other hand have the cis-acting regulatory elements in the promoter and the operator sites.

Question 27

In what order are the structural genes of the lac operon transcribed?

Answer 27

Z, Y, A

The lacZ gene is transcribed first, followed by the lacY and then the lacA, which finally ends with the termination at the terminator sequence denoted as T.

Question 28

What is the function of Lac Z?

Answer 28

codes for beta-galactosidase which breaks down beta-galactoside complex sugar into its component sugars

Question 29

What is the function of Lac Y?

Answer 29

codes for beta-galactoside permease that transports beta-galactosides into the cell

aka: a transporter protein that is needed to transport the complex sugar into the cell.

Question 30

What is the function of Lac A?

Answer 30

codes for beta-galactoside transacetylase, transfers acetyl group from acetyl CoA to beta-galactosides.

Question 31

Explain why mutations in either Lac Z or Y results in bacteria that cannot break down lactose.

Answer 31

beta-galactosidase sugars contain galactose as one of the component sugars. lactose is the most commonly known beta-galactoside. Lactose is made up of glucose and galactose and is cleaved into the monomeric sugars by the action of beta-galactosidase. If mutated, beta-galactosidase cannot cleave lactose

Question 32

Lac A mutants do not display any problems with lactose utilization, what is the reason behind this?

Answer 32

it’s thought that the transacetylase reaction may provide some advantage if the bacteria are grown in the presence of certain analogs of beta-galactosides

Question 33

What is the lac repressor? What gene encodes this repressor?

Answer 33

• A repressor protein that controls transcription of the LacZYA operon. Lac repressor binds to an operator that overlaps the promoter site (site where the RNA polymerase needs to bind and start transcription) at the start of the cluster. & prevents transcription of LacZYA genes
• encoded by the Lac I gene

Question 34

TRUE OR FALSE: The Lac I gene can be located at sites that are nowhere near the lac structural genes and still be functional

Answer 34

TRUE: the Lac I gene had its own promoter and Terminator. so in effect the Lac I gene could actually be located at sites that are nowhere near the lac structural genes and still be functional since the lac I product is diffusible.

Question 35

Mutation in the lac repressor would result in what?

Answer 35

a mutation in this gene would result in continuous expression of the Lac ZYA genes

Question 36

The state in which a gene is expressed continuously is called what?

Answer 36

constitutive expression

Question 37

What are two reasons the lac I repressor protein is in very low abundance in the cells?

Answer 37

1) The lac I gene is an unregulated gene.

however the amount of lac I, is in a way controlled by the fact that the promoter for lac I has a very weak affinity for the RNA polymerase and hence, the transcription of this gene (lac I repressor) is not very great.

2) In addition The mRNA for lac I is also very unstable.

Question 38

In the absences of beta-galactosides, what is the expression levels of the lac operon?

Answer 38

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

Question 39

The Lac operon is negative inducible, what does this mean? And how is it inducible?

Answer 39

it means that it is on the negative control because the repressor protein which is a tetramer prevents expression of the genes.

how is it inducible?

the beta galactoside sugars, which are the substrates of the lac operon, are the inducers, The addition of specific beta-galactosides induces transcription of the three genes in the Lac operon by binding to the Lac repressor inactivating it

Question 40

Why is it that the negative inducible property of the lac operon can be rapidly reversed?

Answer 40

the lac mRNA is extremely unstable and as a result induction can be rapidly reversed

Question 41

TRUE OR FALSE: If bacteria is grown in a medium that lacks beta-galactosides then the bacteria will require the enzyme beta-galactosidase.

Answer 41

FALSE: If bacteria is grown in a medium that lacks beta-galactosides then the bacteria does NOT require the enzyme beta-galactosidase. but the enzyme is still present albeit in very low concentrations.

Question 42

What are basal levels?

Answer 42

the minimum amount /level necessary for function

Question 43

Describe the activity of lac mRNA after induction

Answer 43

At basal levels, lac mRNA is low.

1) Once the inducer is added, transcription of the genes begin immediately and the levels of Messenger RNA increase
2) Then it it levels off (this leveling shows there is a balance between synthesis and degradation of the mRNA.)

3) removal of the inducer returns the Messenger RNA to base the levels.
The lac mRNA is extremely unstable and degrades very quickly, hence the rapid decrease in levels of Messenger RNA following the removal of the inducer.

Question 44

Describe the levels of beta-galactosidase after induction

Answer 44

1) There is basal levels of the enzyme prior to induction
2) following induction there is a lag phase of about 2 minutes during which the transcription is increased but there isn’t a corresponding increase in translation. This is because the protein has to be synthesized.
3) a similar lag is seen between the maximal induced levels of the Messenger RNA and protein. when the inducer is removed no enzyme is synthesized since Messenger RNA is no longer around for translation. however the protein that has already been synthesized is more stable and hence enzyme activity will continue at the induced level much longer.

Question 45

How does an inducer function?

Answer 45

by converting the repressor protein into a form with lower operator affinity

Question 46

The repressor has two binding sites. What are they?

Answer 46

1) one for the operator DNA

2) another for the inducer

Question 47

What are gratuitous induces? Give an example

Answer 47

inducers that resemble authentic inducers of transcription, but are not substrates for the induced enzymes.

Example: IPTG or isopropyl thiogalactoside. it can behave as an inducer but cannot be metabolized by the enzyme

Question 48

TRUE OR FALSE: Inducers are highly specific. In the case of the Lac operon the inducer is not only the molecule that binds to the repressor and induces transcription but it also is the substrate for the induced enzyme

Answer 48

TRUE

Question 49

in the absence of the inducer, the repressor protein takes up what form?

Answer 49

in the absence of the inducer the repressor protein is in the active form and binds to the operator blocking transcription

-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 50

In the presence of an inducer, what will happen to the repressor protein?

Answer 50

in the presence of the inducer, the inducer will bind to the repressor at the inducer site. This binding alters the structure of the DNA binding site such that it lowers the affinity of the operator site for the operator. (This is an example of allosteric control)

From slide: repressor is inactivated by an allosteric interaction in which binding of inducer at its site changes the properties of the DNA-binding site (allosteric control)

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

Question 51

TRUE OR FALSE: The inducer in the case of the Lac operon is lactose

Answer 51

FALSE: The inducer in the case of the Lac operon is NOT lactose but allolactose, which is a byproduct of the lacZ enzyme.

Question 52

Describe how allolactose behaves as a substate for lacZ enzyme

Answer 52

it binds to the Lac repressor and reduces its affinity for the operator. this allows RNA polymerase to now bind to the promoter and begin transcription of the Lac ZYA genes, which get transcribed in the ZYA (lacZ being express first, then lacY, and finally lacA)

Question 53

Fill in the blanks:

Lac Z is needed to make the enzyme that will break down the beta galactoside to _______, for the _______ to induce transcription of the Lac operon. In addition LacY is the transporter that transports the _______ into the cell.

Answer 53

Lac Z is needed to make the enzyme that will break down the beta galactoside to ALLOLACTOSE, for the ALLOLACTOSE to induce transcription of the Lac operon. In addition LacY is the transporter that transports the GALACTOSIDES into the cell.

Question 54

if the operon is in the repressed state, how does the inducer enter the cell? and secondly how did allolactose induce the synthesis of beta galactosidase when the same enzyme is needed to produce the allolactose?

Answer 54

This is addressed in two ways:

1) one due to the basal levels of expression as a certain amount of lacZ and lacY proteins that are always present ,which allows the process to at least get started.
2) Additionally the inducer can be brought into the cell through another optic system. as a result the basal levels of the enzyme produced allolactose, which can then act as an inducer for the Lac operon