Lecture 4- Regulation of Gene Expression in Prokaryotes

Question 1

What is a constitutive gene in comparison to both an inducible and repressor gene?

Answer 1

• Constitutive genes (a.k.a. housekeeping genes) are active all the time. Their proteins help with basic function, vitality, and cell maintenance.
• Inducible/repressible genes are turned on or off depending on the cell’s needs.

Question 2

What is a promoter and name specific promoter region numbers in prokaryotes?

Answer 2

• A promoter is a region of specific sequences that tell the RNA polymerase where to bind.
• (-35) and (-10) are specific consensus sequences. (-10) is known as the Pribnow Box in prokaryotes.

Question 3

What are sigma factors and what do they do?

Answer 3

• Sigma factors are proteins which help initiate transcription in Prokaryotes.
• They help RNA polymerase bind to the promoter.
• There are several types of them
• They interact with different promoters and turn on different genes.

Question 4

What is a cis-operating factor?

Answer 4

• Impact activity of genes on the same DNA molecule.

- All genes have a specific promoter and coding sequence.

Question 5

What is a trans-operating factor?

Answer 5

1. ) Molecules that are made by genes on the main chromosome, but can diffuse over and impact the activity of genes on the plasmid (vise versa).
2. ) Includes molecules that bind to the regulatory sequences:
   a. ) Activator proteins increase sigma factor binding to the promoter region -> helps improve transcription.
   b. ) Repressor proteins decrease sigma factors from binding -> decreases transcription.

Question 6

What is an operon? What are the two types of different operons and describe them?

Answer 6

1. ) An operon has many genes that are influenced by a single promoter sequence. They are transcribed together and all make different proteins.
2. ) An inducible operon is usually off but is turned on when their proteins are needed.
3. ) A repressible operon is usually on but turned off when their proteins are not needed.

Question 7

What are the three areas that make up an operon, describe them? And what is the gene that is associated with the operon and describe it?

Answer 7

1. ) Promoter- region where an RNA polymerase has to bind in order for transcription to occur.
2. ) Operator- region where activators or repressors can bind to in order to increase or decrease transcription.
3. ) Structural genes- code for proteins.
4. ) Regulatory gene- Not found on operon; found a distance away, but it is used to create a protein which turns the operon on or off.

Question 8

What is negative and positive control? Where do these proteins bind? Who makes these proteins?

Answer 8

1. ) Negative control is when a repressor protein binds to the operator and prevents the RNA polymerase from binding to the promoter region. Preventing transcription.
2. ) Positive control is when an activator protein binds next to the promoter region or a distance away and helps the RNA polymerase bind to the promoter. Helps transcription.
   - The regulator gene produces both proteins.

Question 9

What are the four schemes whereby operons work?

Answer 9

1. ) Negative inducible operon: A repressor protein binds to the operator and prevents RNA polymerase from binding.
2. ) Negative repressible operon: A repressor protein is present but is in its inactive form so transcription occurs as normal.
3. ) Positive inducible operon: An activator is present but it is in an inactive form resulting in no transcription.
4. ) Positive repressible operon: An activator is present in its active form and helps RNA polymerase bind to the promoter region.

Question 10

Four types of operons overview: What is negative, positive, repressible, and inducible?

Answer 10

1. ) Negative = repressor is present
2. ) Positive = activator is present.
3. ) Repressible = operon is on.
4. ) Inducible = operon is off.

Question 11

What are the structural genes in a lac operon and what are their functions?

Answer 11

1. ) Lac Z - induces for beta-galactosidase which breaks down lactose into glucose and galactose.
2. ) Lac Y- induces for permease which helps bring lactose into cells
3. ) Lac A - induces for transacetylase: function unknown.

Question 12

What is the LacI gene? Where is it located? Is it a trans or cis acting factore and why?

Answer 12

• LacI gene codes for the LacI repressor which binds to the operator and prevents transcription.
• It is not located in the operon but rather a distance away and that is why it is a trans-acting factor.

Question 13

What are the cis-acting factors in the operon?

Answer 13

Promoter and operator.

Question 14

Why can the Lac Operon be negatively controlled?

Answer 14

• The operon is inducible (it is off unless needed).

- The repressor protein is constitutive and blocking transcription until lactose is present.

Question 15

How does negative control of the Lac Operon work?

Answer 15

• When lactose is present, it is converted into allolactose.
• Allolactose binds to the repressor, which is bound to the operator. Preventing the repressor from binding.
• Transcription occurs and proteins are made.
• When the level of lactose drops, the repressor is able to bind to the operator and prevent transcription again.

Question 16

What is a merozygote?

Answer 16

• A.K.A. a partial diploid cell. It is a cell which takes up a plasmid which contains a copy of its own lac operon.
• Results in two copies of the same lac operon.

Question 17

What is the symbol of a wild-type vs. a mutant type? What do they mean?

Answer 17

• A wild-type has a + symbol and means that the gene is functional.
• A mutant type has a - symbol which means the gene is not functioning.

Question 18

What is conjugation? What type of bacteria does it result in?

Answer 18

• When the donor bacteria gives a plasmid to the recipient bacteria containing a copy of the lac operon.
• Results in a partial diploid merozygote.

Question 19

Mutation Lac Z+ Lac Y- / Lac Z- Lac Y+: What are the results? Are they independent of one another? What type of acting factor is present?

Answer 19

• Although there are two non-functional copies of Lac Z and Lac Y genes, there are two functioning genes.
• Protein production was not hindered, beta-galactosidase and permease were present.
• One working copy allowed for lactose to be metabolized.
• A cis-acting factor is present. One mutant copy in the main chromosome or plasmid did not affect production.

Question 20

How are Lac Z, Lac Y, and Lac A genes not independent of one another?

Answer 20

• If something stops translation of the Lac Z gene, then translation for the Lac Y and Lac A genes does not occur.
• Ex.) Nonsense mutation

Question 21

Mutation Lac Z- Lac I+ / Lac I- Lac Z+: What are the results? Are they independent of one another? What type of acting factor is present?

Answer 21

• The repressor on the main chromosome from the functioning Lac I gene diffuses over to block the Lac Z gene on the plasmid from being translated.
• Lactose still needs to be present in order for the functioning Lac Z gene to be translated.
• This is a trans-acting factor because the repressor from the main chromosome diffuses to the plasmid to block translation of Lac Z when lactose is not present.

Question 22

Mutation Lac Z+ Lac I s / Lac I- Lac Z+: What are the results? Are they independent of one another? What type of acting factor is present?

Answer 22

• This is a superrepressor for the Lac I gene.
• This prevents the inducer (allolactose) from binding to the repressor and preventing it from binding to the operator, preventing transcription.
• Even when lactose is present, the lac operon is never on.
• This is also a trans-acting factor because the Lac I s diffuses to the plasmid and blocks that operon from transcribing

Question 23

Mutation Lac Oc Lac Z+ / Lac O+ Lac Z+: What are the results? Are they independent of one another? What type of acting factor is present?

Answer 23

• Consitutive mutation in the operator.
• The operator does not let the repressor bind which ultimately leads to transcription all the time and the presence of betagalactosidase even when lactose is not present.

Question 24

Mutation Lac Oc Lac Z- Lac I+ / Lac O+ Lac Z+ Lac I+: What are the results? Are they independent of one another? What type of acting factor is present?

Answer 24

• Beta galactosidase will only be produced when lactose is present because the Lac Z gene in the main chromosome is non-functioning.
• The Lac Oc only works on the same chromosome meaning that this is a cis-acting factor.

Question 25

Mutation Lac P+ Lac I+ Lac Z+ /Lac P- Lac I+ Lac Z+: What are the results? Are they independent of one another? What type of acting factor is present?

Answer 25

• The promoter region mutation where it does not let RNA polymerase to bind is a cis-acting factor.
• Beta galactosidase is formed when lactose is present due to the functioning of the main chromsome.

Question 26

What is the positive control of the lac operon? What is it called and how does it work?

Answer 26

• A.K.A. catabolite repression.
• Cell would rather use glucos than lactose for its energy source, so it shuts down the lac operon.
• There is an activator binding site upstream from the lac operon.
• The activator binding protein is cyclic AMP of CRP, but it needs to be binded with cAMP in order to become active and bind to the activator site.
• When glucose is present, cAMP is not created due to the fact that glucose inhibits adenylate cyclase (which makes cAMP). This does not allow for CRP-cAMP complexes, which in turn stops transcription.
• When glucose concentration lowers, cAMP is made and the CRP-cAMP complex can bind to the activator site leading to the operon functioning normal again.

Question 27

How does the trp operon work? And what type of operon is this?

Answer 27

• trp operon has five structual genes that code to creat the amino acid tryptophan.
• The trp operon is always on unless tryptophan is not needed.
• The regulatory gene creates a repressor protein in its inactive form. Tryptophan attaches to the repressor protein to active it (called the corepressor), which then binds to the operator of trp operon.
• Once levels of trypotphan diminish, the repressor becomes in-active again and the trp operon transcribes again.

Question 28

Can multiple operons be controlled by the same repressor/activator? What is this called?

Answer 28

• Activators and repressors acting as trans factors diffuse throughout the nucleoid and can regulate many different genes as long as the operator and promoter sequences fit these activtors and repressors.
• A regulon is what you call a group of genes that are regulated by a single activator/repressor.

Question 29

What is the leader gene? Where is it located? Why is it important?

Answer 29

• The leader gene is found on the trp Operon between the operator and the five structural genes.
• RNA polymerase transcribes the leader gene and then the ribosome translates it into the leader peptide.
• This determines whether the structural genes get translated into tryptophan.

Question 30

How many regions of the leader gene are there and what are their purposes?

Answer 30

• There are four regions.
• Region 1 has two coding regions for the amino acid tryptophan.
• Region 3 can bind to either region 2 or region 4 but not both. These regions form a stem and loop.
• RNA polymerase binds right after region 4.
• If regions 3 and 4 form the stem-loop than this blocks RNA polymerase from binding leading to no transcription of the structural genes.
• If regions 2 and 3 bind then transcription does occur.

Question 31

What happens to the leader gene in the trp operon if there are high levels of tryptophan?

Answer 31

• If there are high levels of tryptophan, then the ribosome has no problem reading the tryptophan because there are plenty tryptophan- tRNAs and can go all the way to the STOP codon in region 2.
• This blocks the formation of the stem and loop in region 2 which means the stem and loop binds to region 3 and 4 stopping transcription of the structural genes.

Question 32

What happens to the leader gene in the trp operon if there are low levels of tryptophan?

Answer 32

• If there are low levels of tryptophan, then the ribosome has trouble translating the leader gene into the leader peptide because it cannot find enough tryptophan tRNAs.
• The ribosome does not go all the way to the STOP codon resulting in the stem and loop forming at regions 2 and 3.
• Region 4 is left open and RNA polymerase is able to bind to the promoter region and transcribe the five structural genes.

Question 33

How does the transcription factor TrmBL1 activate and repress transcription?

Answer 33

• TrmBL1 simultaneously represses genes that synthesize transport proteins for other sugars, while activating glucogenesis.
  1. ) TrmBL1 protein binds to a site upstream from the B recognition element and TATA box for maltodextrin and maltose. This does not allow RNA polymerase to bind which in turn leads to no transcription.
  2. ) TrmBL1 protein also attaches to a site upstream from the B recognition element and TATA box where genes encode enzymes for glucose synthesis ad recruits TBP, TFB, and RNA polymerase to the site, activating transcription.

Question 34

What activates transcription factors and how does it work?

Answer 34

• Signal Transduction activates transcription factors.
  Mechanism:
  1.) Molecules from the environment bind to extracellular transmembrane sensory kinase proteins which undergo self-phosphorylation.
  2.) The phosphate group travels to the response regulator which is the transcription factor.
  3.) The activated response regulator then goes and represses/activates its specific gene.

Question 35

How do bacteria activate genes through quorum sensing?

Answer 35

• Bacteria secrete and activator known as AHL which activates a gene that produces luciferase. (Makes bacteria glow)
• If there are not enough bacteria than AHL cannot diffuse into the bacteria cell, but when enough bacteria accumulate then AHL can diffuse into the cell and activate luciferase.

Question 36

What is the stringent response and why does it happen?

Answer 36

• The stringent response occurs when nutrients in the environment are low.
• This triggers the bacteria cell to stop ribosomal, tRNA, rRNA, DNA, and protein synthesis.
• This triggers the synthesis of more amino acids so new proteins can get synthesized to adapt to the new environment.

Question 37

How does Anti-sense RNA impact translation?

Answer 37

• Anti-sense RNA have sequences that bind to mRNA and have the ability to decrease or increase translation.

Question 38

What are riboswitches and what do they do?

Answer 38

• Riboswitches are proteins or other molecules that bind to RNA and dictate if translation occurs or not.

Question 39

What are ribozymes?

Answer 39

• RNAs that bind to mRNA and signal the mRNA to cleave themselves. They act like enzymes.