Lecture 14

Question 1

What is the difference between constitutive and regulated transcription?

Answer 1

constitutive expression: some genes in bacteria is expressed no matter the environment ( e.g: housekeeping genes)

regulated transcription: genes are turned on depending on environment

two methods of regulated transcription
- regulation of transcription initiation (on/off)
- regulation of the amount of transcription

Question 2

What is one advantage and one disadvantage of regulating gene expression?

Answer 2

Pro: save energy

Con: requires accurate sensing of the environment

Question 3

What is a repressor protein? What is its allosteric site?

Answer 3

repressor: bind DNA to block rna polymerase from transcribing gene

allosteric site: location on repressor where another compound is bound or unbound and affects whether the DNA binding domain of the repressor can bind to the DNA

Question 4

What is an inducer? What is a corepressor?

Answer 4

• inducer can bind repressor and release DNA to allow transcription
• corepressor binds repressor and induce DNA binding ( allows repressor to bind DNA)

trp repressor

Question 5

What is an activator protein?

Answer 5

have an allosteric domain where an effector or inhibitor can bind and affect whether the DNA binding domain of the activator will bind DNA and promote transcription or not.

help w/ transcription but not necessary

Question 6

What is an effector? What is an allosteric inhibitor?

Answer 6

EFFECTOR COMPOUND: binds to activator to allow it to bind DNA and upregulate transcription

allosteric inhibitor: bind to activator and prevent successful binding of activator to DNA this inhibiting transcription

Question 7

What are operons? What are two advantages of operons?

Answer 7

bacterial genomes organized into groups of co-regulated genes

1. efficiency
2. keep the genome compact

Question 8

What conditions trigger expression of the lac operon? What conditions prevent expression in the
lac operon?

Answer 8

Lactose present and Glucose absent: Lac Operon is on
Glucose Present or Absent /Latose Absent : Lac Operon is off

Lac Repressor: constitutively expressed( always on). repressor binds to operator sequence such that rna polymerase does not transcribe

In presence of lactose ,
1. permease imports lactose into the cell

2. lactose conv to Allolactose by Beta-Gal
3. lacA: protects the cell from damaging byproducts of lactose
4. the allosteric domain of the repressor binds allolactose( inducer) and the repressor releases DNA to allow for transcription

Question 9

What is a polycistronic mRNA?

Answer 9

One mRna molecule that encodes multiple proteins and each portions of the mRna have their own start and stop codons

Question 10

In the lac operon: What is the operator? What binds it? What is the consequence of this? When is
it bound?

Answer 10

the operator is a dna sequence upstream known as lacO, rna polymerase binds it if lactose is present

when lactose is unavailable repressor binds to the operator(lacO) and inhibits transcription

Question 11

In the lac operon, what binds the repressor protein and what is the consequence of this?

Answer 11

allolactose is an inducer that binds repressor and releases DNA from reprepressor to allow for prescription

Question 12

How is allolactose formed? How does lactose enter the cell if the operon is off?

Answer 12

allolactose is formed by the breaking down of lactose by beta-gal

lactose enters the cell with the help of permease

there is always some amount of permease/ beta -gal allowing some lactose to enter the cell –> be broken down into allolactose and de-repressed the lac operon

this faulty repression/ leaky expression allows for function of operon if repression were 100% there would be no way to turn the operon on again

Question 13

When does cAMP bind CAP? What is the consequence of this?

Answer 13

when there is no glucose present in the cell

cyclic amp is produce in high levels from the conversion of ATP to cAMP with the help of adenylate cyclase

cAMP then binds a protein called CAP which (turns on lac operon) binds DNA and helps rna poylmerase to bind DNA and transcribe lac operon to produce energy

Question 14

What is the difference between basal and leaky expression?

Answer 14

leaky expression: faulty repressor binding that can be reversible ( very little transcription even in the absence of the inducer(e.g absence of allolactose))

basal expression: lack of repression + lack of activation due to lack of activator ( while RNA polymerase can bind activator can not facilitate its movement as much so very little transcription occurs)

e.g: lactose +glucose present

lack of activation: glucose present–> adenylate cyclase does NOT convert ATP to cAMP–> cAMP not binding CAP–> CAP not binding do DNA –> RNA polymerase movement not facilitate so little transcription

lack of repression: lactose conv. to allolactose–> binds to repressor–> repressor dissociates–> no repression

Question 15

What types of mutations lead to constitutive mutants? Which act in cis? Which act in trans?

Answer 15

expressing operon even when glucose is present and lactose is absent ( uncontrolled/undesired transcription)

cis-acting operator mutation: these mutations only affect genes on the same chromosome and result in mutation in the operator sequence that prevents the repressor from binding the operator

transacting: mutation within the repressor sequence that prevents repressor protein from binding to the operator.

affects genes on both chromosomes

Question 16

What types of mutations lead to non-inducible mutants? Do they act in cis or in trans?

Answer 16

Super-repressor mutations are noninducible as they lead to a mutation in the repressor protein that prevents it from binding inducer (allolactose) CAUSING the repressor to always be bound.

These are trans-acting I^S mutations.

Question 17

Define negative feedback

Answer 17

negative feedback is when the abundance of a protein or substance results in. signal being sent to stop production of said protein

Question 18

Contrast typical characteristics of catabolic and anabolic operons.

Answer 18

catabolic operons: inducible by the presence of a particular nutrient, breaking down large molecules into smaller ones (yields energy)

e.g: lac operon ( breaking down lactose to get glucose for energy)

anabolic operons: repressible by negative feedback .if there is abundance of end product transcription is stopped; constructing large molecules from smaller ones (requires energy)

Question 19

What is attenuation? What is an advantage of attenuation compared to a simple on/off
mechanism?

Answer 19

the degree of expression is tightly controlled –> expression is being dimmed instead of switched on or off this is important in operons involved in production of amino acids as cells have evolved to have constant levels of amino acids so suddenly switching it off could have adverse effects

Question 20

What is the function of the 1-2 stem loop?

Answer 20

The 1-2 stemp loop sows transcription down such that a ribosome can bind the trpL mrna.
If this ribosome does not occur 1-2 and 3-4 can occur simultaneously eliciting transcription termination.

Question 21

What determines whether a 2-3 or a 3-4 stem loop will occur?

Answer 21

the 2-3 stem loop prevents termination from happening and the polysitronic mrna need to make tryptophan is created.

What determines: attenuation of how the trpL mrna is being translated.

there is a 14 AA codon that includes 2 consecutive trp codons.

if there is a lot of trp there will be more 3-4 stem loops as we do not need to produce trp.

So if there is a lot of trp the ribosome bound to the trpl mRNA will translate fast . Once the ribosome overlap with region 2, the 2-3 stem loop cannot form so only the 3-4 stem loops will form.

if there is not a lot of trp we will make more 2-3 stem loops. The ribosome will stall/ translate slowly due to there being too little tryptophan and will not occupy region 2, so the 2-3 stem-loop will form preventing 3-4 stem-loop from being created.

Question 22

Suppose the trp operon had its attenuator codons mutated to stop codons, what would be the
expected result?

Answer 22

If there is high tryptophan it would prevent the ribosome from reaching region 2 so you would always have constitutive expression of the trP operon-

Question 23

Explain how alternative sigma factors can give rise to genome wide alterations in gene
expression.

Answer 23

when you increase expression of an alternative sigma factor under certain environmental conditons, promoters of different genes specific to that sigma factor are recognized and a bunch of genes specific to those promoters are turned on.

increase in sigma 32 as opposed to sigma 70 in e.coli recognizes on heat shock promoters which turns on the heat shock genes.

Question 24

Explain how translational regulation for ribosomal proteins functions. Is this negative feedback?
Why or why not?

Answer 24

1. protein binding mRNA ( e.g ribosomal proteins that are transcribed by operons) can obscure its own shine delgarno promoter sequence and prevent it from being translated. So if you translate a lot of ribosomal proteins the abundance of them will provide negative feedback by binding its own mrna.

Question 25

Explain how antisense RNAs can block translation.

Answer 25

antisense RNA: RNA that is complementary to the shine delgarno region of MRNA.

IS10 gene transcribes transposase (jumping gene that causes mutation that causes null alleles).

Has two promoters oriented in opposite directions(bidrectional promoters) P-In is a weak promoters that drives expression of transposase, P-out stronger promoter that encodes antisense RNA . The RNA is base paired with the antisense RNA so the shin delgarno sequence is obscured so none of the RNA is translated.

Question 26

Compare and contrast tryptophan’s role as a co-repressor in the trp operon and GlcN6P’s role in
regulating glmS. How are these mechanisms similar? How are they different?

Answer 26

tryptophan is a co=-repressor that when bound to repressor allows repressor to bind DNA and prevent transcription

when tryptophan is absent there will be transcription of the trp operon

glmS catalyzes the formation of sugar glcN6P

high glcN6P= glcN6P bind stem loops and 5’ end of mRNA is cleaved. RNAS J1 degrades the 3’ end.

low glcN6p: mRNA is translated

so negative feedback is common for both but the mechanism is different