Test1: Lect5 Peter Zuber

Question 1

One gene one enzyme theory:

Answer 1

No notion of gene regulation, all genes transcribed

Question 2

What does EMB agar + methylene blue + eosin yellow do?

- Nomenclature?

Answer 2

It assays for sugar fermentation, which will give colored colonies.
- Nomenclature?
Lac+ (lactose fermented)
Lac- (lactose not fermented)

Question 3

Monod and yudkin performed what experiments?

Answer 3

They found in the presence of 2 sugars, bacteria grew, plateaued, and then grew again

Question 4

How did yudkin interpret the grow plateau grow?

Answer 4

Adaptive enzymes: the enzymes only became active in presence of their substrate

Question 5

What finding of monod upturned yudkin’s adaptive enzyme explanation?

Answer 5

1: The actual levels of the protein were rising.
2: Beta galactosidase binds but does not induce, it also reacts with its own enzyme, but doesn’t induce.

Question 6

Define Operon:

Answer 6

a functioning unit of DNA containing a cluster of genes under the control of a single promoter

Question 7

LacZ:

Answer 7

beta-galactosidase, its lactase cuts lactose to glucose and galactose

Question 8

LacY:

Answer 8

Lactose permease, allows entry of lactose into the cell

Question 9

LacA:

Answer 9

Galactoside transacetylase (does not appear to be necessary for lactose catabolism)

Question 10

LacI:

Answer 10

Encodes a repressor which binds to the operator of the lac operon in the absence of lactose.

Question 11

How was the operon theory tested/developed?

Answer 11

A mutant was found which could grow on beta galactosidase. Beta galactosidase does not induce the lac operon, but a lacI mutant was selected for.
This showed that one could have a mutant in induction in a separate gene, so that’s gene product must be involved in induction. Thus the operon hypothesis.

Question 12

Operons exist in which the control site is not an operon, explain?

Answer 12

The control site is likely the promoter. The repressor which binds to the promoter’s site.

Question 13

Order of lac operon

Answer 13

LacI (repressor) -> promoter (RNA pol binds here) -> operator (LacI binds here) -> LacZ -> LacY -> LacA

Question 14

LacI-:

Answer 14

Produces repressor which cannot bind the operator.

Operon will be constitutively active

Question 15

LacIs:

Answer 15

1: Produces an repressor which does not release the operator
2: it cannot bind lactose to induce release
3: this is a gain of function mutation

Question 16

LacO:

Answer 16

The operator site

Question 17

LacOc:

Answer 17

1: Does not bind the repressor as well, constant activity.
2: trans acting (operator is clearly not diffusible)
3: dominant to LacIs

Question 18

What experiment showed that LacI is a trans acting inhibitor not a localized inducer?

Answer 18

Conjugation experiment
Bacteria with Hfr (F+) lacIOZYA Str(s) T(6)
+
Bacteria with
Hfr (F-) lacI- lacZ- Str(-) T(-)
=
In presence of streptomycin and phage, lactose still produced. Means the plasmid can make repressor which acts on chromosome. Trans acting.

Question 19

Does LacI have its own promoter?

Answer 19

Yes, it is always active.

Question 20

Trp (tryptophan):

• Talk about its synthesis:
• Fast adaption method:
• Slow adaption method:

Answer 20

• Talk about its synthesis:
  Synthesized only when the cell doesn’t already have it (energy efficiency)
• Fast adaption method:
  Feedback inhibition of trp on the 1st enzyme
• Slow adaption method:

Question 21

Steps for analysis of regulation:

Answer 21

1: examine expression
2: Isolate mutants
3: analyze mutants (what can they do and can they not do, what genes were changed)
4: formulate hypothesis (I think this gene is the operator, I see continuous activity when its gone, and I have reason to believe there is a repressor)
5: Analyze in vitro (replication in vitro, outside of cell, with repressor or activator, and a DNA template with your operon, measure p32 incorporation)
6: revise models

Question 22

How was Trp mutant selected for?

Answer 22

Grown in a form of tryptophan which was toxic when not processed. Mutants which survived produced trp processing enzymes constitutively.

Question 23

Positive control of operon:

Answer 23

Has an inducer as regulatory protein

Question 24

Negative transcription control:

Answer 24

Uses a repressor to stop transcription (both trp and lac regulation is negative)

Question 25

What two types of negative regulation are seen? Example of each.

Answer 25

Presence of substrate induces transcription (binds to and disables repressor)
- Lac operon
Presence of substrate inhibits transcription (binds to and activates repressor)
- Trp operon

Question 26

Trp operon:

Answer 26

negative t

Question 27

Lac operon, lactose acts as a:

Answer 27

Inducer (removes the impressor)

Question 28

Trp operon, trp acts as a:

Answer 28

Corepressor (binds to repressor, helps it repress)

Question 29

Trp operon:

Answer 29

Same basic flow as lac operon, only difference is repressor has a corepressor trp.

Question 30

Lambda phage:

- Lifecycle options:

Answer 30

• Lifecycle options:
  Lytic (kills cell)
  Lysogenic (lives in cell)

Question 31

Regulation of the lambda phage:

Answer 31

Protein CI (C1). Acts a repressor on two operons which inhibit lytic state.
One of these operators it also acts as an activator

Question 32

TrpR:

Answer 32

Trp repressor. Trp runs on a corepressor, so it uses TrpR not a TrpI

Question 33

Describe lambda operons:

Answer 33

1: Has two operons. Genes are OL1, OL2, OL3 in one and OR1, OR2, OR3 in another. OR operon is also near C1 gene, and C1 bound at this operator induces C1 formation. C1 is the repressor for both of these.
RecA is an inducer for OR and OL, and allows transcription which results in lysis.

Question 34

Ind-:

Answer 34

Mutant, were C1 can no longer bind RecA and be activated

Question 35

Default state:

• Define:
• Positive vs Negative control?

Answer 35

• Define:
  if the operon is placed in vivo, with nothing but RNA polymerase and nucleotides, does it produce mRNA or not? That is the default state.
• Positive vs Negative control?
  Positive -> Default state no or little mRNA
  Negative -> Default state mRNA produced

Question 36

Describe the Ara operon:

Answer 36

AraI is the operator next to promoter.
AraO is an operator away from the promoter.
If arabinose is present, AraC (a repressor and activator) binds to just araI, activating the polymerase.
If arabinose is absent, AraC binds to both araI and araO, bends the DNA which represses the polymerase.

Question 37

What two types of positive regulation are seen? Example of each.

Answer 37

1: Activator (activated by inducer), activator binds when ligand (inducer) present.
- Ara
2: Activator (deactivated by corepressor), activator binds when corepressor is absent.

Question 38

Two component system:

Answer 38

A membrane sensor kinase is phosphorylated by events outside the cell. It in turn phosphorylates the response regulator (which is either an activator or repressor)

Question 39

The glucose effect:

• Define:
• This is an example of what?

Answer 39

• Define:
  When glucose is present, transcription of all other alternative carbon source enzymes is halted
• This is an example of what?
  Catabolite repression

Question 40

Catabolite repression:

• Define:
• Explain it in the lac operon:

Answer 40

• Define:
  When preferred carbon source is there, enzymes which allow breakdown of other carbon sources is shut down.
• Explain it in the lac operon:
  CAP is an activator of the lac operon (positive control). cAMP is the inducer. cAMP is present when glucose is not. Transcription takes place when glucose is not present.

Question 41

Sutherland theorized cAMP was cause, how did he find mutants to test this?

Answer 41

Grew e-coli on EMB with many different sugars. Was looking for white colonies (cannot ferment sugar even in absence of glucose)

Question 42

What two mutants did Sutherland find in his study of cAMP?

Answer 42

1: cya gene mutant: cell cannot grow on any sugar but glucose, without cAMP being in medium
- This is a adenylyl cyclase mutant, it cannot form cAMP
2: crp gene mutant: cell cannot use any sugar but glucose
- This is a CAP mutant, the activator is just not working.

Question 43

Why is cAMP low when glucose is high?

Answer 43

moving glucose across phosphorylates a protein which inactivates adenylate cyclase

Question 44

What type of sequence does CAP-cAMP bind?

What effect does its binding have?

Answer 44

a palindromic sequence

Causes a bend in the DNA strand

Question 45

Where does CAP bind in the Lac operator?

Answer 45

right before the promoter

Question 46

Two functions of the alpha protein in RNA pol?

Answer 46

Recognizes the promoter sequence

The c-terminal domain (CTD) of the alpha protein interactions with transcription factors (like CAP - cAMP)

Question 47

How does CAP act in arabinose operator?

Answer 47

It binds and straightens the DNA segment. Still requires arabinose to be straight however, so -glucose +arabinose = transcription.

Question 48

Study on CAP-cAMP as global regulator:

• Process:
• Findings:

Answer 48

- Process:
Let CAP bind to gene ->
formaldehyde to hold it there ->
Cut up genome ->
Immuno precipitate protein ->
Chip hybridization (to see which sequences you have.
- Findings:
It is indeed in many places.

Question 49

Experiment finding the protein interface using genetics:

Answer 49

1: Found the consensus sequence for CAP.
2: selected for Lac- (lac always off) which had a mutation in the CAP sequence, CAP cannot bind
3: Find mutant CAP which can bind to mutated Lac-, and activate it (this mutated CAP will only be able to bind to that one domain)
4: Other CAP sugars allows entrance of phosphomycin, so phosphomycin was used to select for the protein
5: Proved mutation was in CRP gene (CRP produces CAP) by using homologous recombination to float out just CRP and place it in another cell using the lambda phage. If it restores Lac+ then the modified CAP is enough to fix it
6: looked at where modification in gene occurred, and predicted the amino acid number and amino acid. He was right! YAY!