Lecture 3 - Repressor and Operator

Question 1

What is the o gene?

Answer 1

operator for the Lactose operon (z, y, a genes)

Question 2

Who discovered the Lac operon?

Answer 2

Fracois Jacob, Jacques Monod André Lwoff

Jacob and Monod

Question 3

What special properties did Monod predict for the i-Repressor protein?

Answer 3

Allosteric - 2 different binding sites (for operator, inducer)

Undergo conformational change from binding the inducer, resulting in the inability to bind the operator

More than 1 subunit (oligomeric) so change in shape is cooperative: one subunit binding an inducer makes it easier for remaining subunits to bind inducers

Question 4

What is the R form and T form of the i-Repressor protein?

Answer 4

R form - Relaxed. Active conformation to bind operator DNA

T form - Tense. Inactive conformation for operator DNA binding.

Question 5

Who purified and characterized the lac operon?

Answer 5

Wally Gilber and Benno Muller-Hill

i(q) introduced by using phase transduction

Question 6

How is the i-repressor protein isolated?

Answer 6

Using i(q). These have the wt structure but in abundance.

Question 7

Outline the structure of the i-Repressor protein

Answer 7

Tetramer

Mr = 4 x 37,000

Question 8

How is the affinity of i-Repressor protein measured?

Answer 8

Equilibrium dialysis or Gel filtration

Giving binding constants Kd(dissociation) or Ka(association)

Kd = 1/Ka

Kd(operator) is x1000 when inducer is present

Question 9

How is the binding site of i-Repressor found? Give details

Answer 9

Protection and footprinting assay.
Operator site has two-fold(dyad) symmetry: palindromic sequence

Symmetrical DNA binding site is recognised by a symmetrical i-Repressor protein

Question 10

Does the promoter and operator region of lac operon overlap?

Answer 10

YES

Question 11

How fast does the lac operon respond in the presence of an inducer?

Answer 11

1-2 mins time lagg

Change in B-Galactosidase is proportional to conc. of its mRNA

Question 12

Define operon

Answer 12

A cluster of structural genes downstram from a regulatory region of DNA. Expression of the structural genes is controlled by the regulatory region and gene products are related in function

Question 13

Define negative control circuits

Answer 13

Active form of the repressor proteins turns off transcription by binding to the operator DNA and blocking access of RNA polymerase beyond promoter.

Question 14

Define catabolic and anabolic enzymes

Answer 14

catabolic : enzymes that are induced by the small molecules that they are going to degrade i.e lac operon

anabolic : enzymes that are repressed by small molecules (corepressors) that they have already biosynthesized. i.e tryp operon

Question 15

What are the gene pathways opposite to lac operon?

Answer 15

Biosynthetic pathway of tryptophan.
Production of tryptophan is stopped when tryptophan is present
Tryptophan is a corepressor

Question 16

What is the glucose effect (Diauxic growth)?

Answer 16

When 2 metabolites is present. One is more preferred than than the other.

e.g Glucose is utilized first, when levels fall, cAMP increases, which induces the lac operon to metabolise lactose.

Question 17

What are Catabolite activator protein (CAP)

or cAMP receptor protein (CRP)?

Answer 17

Protein which activates the promoter of the lac operon

its a dimer of Mr = 2 x 22,500

Two-fold rational symetry like the lac operator

Question 18

What is the effect of CRP binding?

Answer 18

DNA bending

Question 19

How is the CRP regulated?

Answer 19

By glucose

Low glucose = low carbon catabolite = low ATP = increased cAMP

cAMP + CAP -> cAMP-CAP complex -> Increase transcription by RNA polymerase x50

This is a POSITIVE CONTROL CIRCUIT: active form of regulator protein+small molecule turns on transcription

cAMP is the inducer
CAP is the apoinducer (naturally inactive)
cAMP-CAP is the inducer-apoinducer complex (active)

Question 20

What is the CAP binding site mutations?

Answer 20

Lac L8 : GC to AT
Lac L29 : CG to TA
These are mutations which decrease, but do not completely abolish the affinity for the CAP protein.

Question 21

Where is the CAP binding site found?

Answer 21

Within the promoter region of the lac operon

Question 22

What is the promoter mutation?

Answer 22

Lac p(r)la : CG to AT
Lac UV5 : CG to AT and TA to AT
These are mutations which increase the strength of the lac promoter. UV5 mutants in the “-10 box” has reverted to the optimal E.coli consensus sequence.

Question 23

What is the Inducer exclusion mechanism?

Answer 23

Mediated by the phosphotransferase transport system (PTS)

PTS is constitutively expressed which is a set of membrane proteins which transports glucose into the cells. When glucose is present, PTS is active. PTS inhibits lac permease to prevent uptake of lactose so operon cannot be induced.

Question 24

What is the adenylate cyclase modulation mechanism?

Answer 24

Mediated by the phosphotransferase transport system (PTS)

Adenylate cyclase is a PTS component, called E II A(glc).
It is PHOSPOHRYLATED in ABSENCE of GLUCOSE.

The phosphorylated protein activates adenylate cyclase to make cAMP, which then activates CAP to enhance transcription

Question 25

What is Blue/White screening?

Answer 25

A method to indicate successful cloning of a DNA insert in a vector. Recombinant DNA fragment is inserted to vector via. restriction enzyme. successful insertion disrupts coding sequence so that no B-galactosidase is made, making white colonies.

Question 26

How is Blue/White screening optimized?

Answer 26

Lac UV5 - galactosidase expression
Lac L8 -reduce leaky
Lac i(q) -reduce leaky
IPTG - induction