Regulation of Gene Expression

Question 1

Lactose

Answer 1

The Prokaryotes use Lactose after glucose is used up

Lactose –> Galactose + Glucose by the enzyme B-Galactosidase

Question 2

Basic Principles

Answer 2

1. Functionally related genes in prokaryotes are organized into operons
2. The genes of an operon are coordinately controlled
3. Regulation is exerted primarily at the level of transcription
4. At least three genetic regulatory elemets are required to exert transcriptional control
   
   1. A regulatory gene (LacI gene codes for repressor protein)
   2. A promoter (nucleotide sequence that serves as recognition motif for RNA P binding)
   3. An operator: nucleotide sequence that binds repress protein
5. Inducers bind to the repressor to weaken affifinity for operator
   
   1. In Lac operson, inducers are allolactose (side product of B-galactosidase)
6. Lac repressors binds tightly to the operator as a tetramer dimer config

Question 3

Negative Regulation

Answer 3

Negative regulation invovles repressors:

• Repressor binds to DNA and shuts down transcription
• Signal causes repressor to dissociate from DNA –> transcription induced

Question 4

Positive Regulation

Answer 4

Positive regulation involves activators

• Molecular signal causes dissociation of activator from DNA -> inhibits transcription
• Molecular signal causes binding of activator to DNA, inducing transcription

Question 5

After Glucose is used up

Answer 5

When Glucose levels decreases, cAMP levels increase:

• cAMP binds to CAP (Catabolite Activator Protein)
• cAMP-CAP binds to promoter, binding facilitates RNAP binding to promoter
  
  • stabilizes RNA P and creates additonal sities for interaction

*When glucose is absent, lac operon transcription is stimulated by CRP-cAMP

Question 6

When lactose is absent

Answer 6

If lactose is absent –> repressor stays bound -> no transcription even when CRP-cAMP is bound

Regardless if Glucose is high and cAMP is low

or

Glcuose is low and cAMP is high

No Lactose - no gene expresson

• Allolactose is molecular signal that dissociates repressor however with no lactose present = no allolactose

Question 7

When Lactose is present, transcription depends on Glucose level

1. Glucose high, cAMP low, lactose present
2. Glucose low, cAMP high, lactose present

Answer 7

Glucose high, cAMP low, lactose present:

• With lactose present, allolactose will be present, but because of only low cAMP levels, only low level of gene expression

Glucose low, cAMP high, lactose present

• Allolactose can reduce affinity of lacI repressor for operon and cAMP binds to CRP to stabilize RNA P to cause high level of gene expression

Question 8

Two requirement for strongest induction of the lac Operon

Answer 8

1. Lactose must be present to form allolactose to bind to repressor and cause it to dissociate from operator (reducing repression)
2. [Glucose] must be low so that cAMP can increase, bind to CRP, and the complex can bind near the promoter (causing activation)

Question 9

Summary of Glucose Effect

Answer 9

Question 10

Gene Regulation in Eurkaryotes

Answer 10

Basal Trascriptional Machinery:

• binds to core promoter
• composed of RNA Pol II and general transcription factors (TATA binding protein)

Regulating Proteins

• Activator and Repressors
• bind to enhancer sequences or silencer sequences, act at a distance

Coactivators (mediators)

• Link the basal transcriptional appartus to upstream regulatory proteins

Insulators

• Define regulatory regions of DNA

Question 11

Ordered assembly of the basal transcription factors

Answer 11

1. Order assembly starting with the TATA binding protein
2. Recruits Pol II - assembly of Preinitiation Complex (PIC)
   
   1. Key rate determining step (Rate of transcription)
3. Transcription bubble forms and DNA unwinds
4. Transcription initiates and mRNA elongation
5. Transcription terminates when CTD is dephosphorylated
   
   1. facilitated by termination factors

* Assembly of PIC is the rate determining step of eukaryotic transcription

Enhancers and Enhancer binding proteins increase rate of PIC Formaiton

Question 12

Transcription Factors (upstream activator factors)

Answer 12

Contain discrete functional domain:

1. DNA binding
2. Factor dimerization
3. Transcription activation

Classes of DNA binding proteins

1. Helix turn Helix
2. Zinc finger
3. Leucine zipper
4. Helix loop helix

Question 13

How do transcription factors regulate gene expresson

Answer 13

While one domain of a transcription factor binds to DNA, other domains are involved in protein-protein or ligand interactions.

Activation domains:

• Acid domains
• Glutamine-rich domains
• Proline-rich domains

All these stabilize co-activator proteins

Question 14

Essential Concepts

Answer 14

1. Promoter upstream of gene and often linked to activating sequences called enhancers
2. Transcription can be activated or repressed in response to cellular signals
3. Activation domains interact with the basal transcription apparatus to stimulate transcriptional activation.