Regulation of gene expression

 

A. Introduction

  1. Need for regulation
  2. Types of regulation
    1. negative regulation involves a repressor protein
    2. positive regulation involves an activator protein

B. The lac operon as a paradigm of negative regulation

  1. diauxie was first evidence of de-repression
  2. two genes -lacZ and lacY, and their products, beta-galactosidase and permease, were shown to be induced by lactose
  3. constitutive mutants, that showed unregulated levels of these enzymes, were isolated
    1. constitutive in this context means that the enzyme levels were always high, whether or not lactose was present
    2. remember that this is a mutant phenotype (and genotype). The original notation was to call the wild-type, inducible genotype i+ and the constitutive mutant i-. 
    3. today, we use the term constitutive to mean "unregulated" (and it doesn't imply a mutant genotype).

 

Strain 

Phenotype 

i+Z+Y+ 

lacZ and lacY are inducible 

i-Z+Y+ 

lacZ and Z are constitutive 

i+Z+Y+/ i-Z-Y- 

inducible (so, i- is recessive, see?) 

C. Gratuitous inducers were synthesized by Lederberg

  1. these are chemical analogs of lactose that can induce the lac operon, but are not metabolized by the cell.
  2. examples, IPTG (isopropyl thiogalactoside) and TMG (thiomethyl galactoside).
  3. They were useful to study the induction process without complications caused by the metabolism of the inducer.

D. PaJaMo (Pardee, Jacob, Monod; 1959)

  1. performed a series of matings (conjugations) with strains of E. coli that contained different alleles of lacI and lacZ.
  2. one of the most informative matings was: F'i+z+/i-z-
    1. with this type of mating, you can actually do a time course
    2. two time courses, one with inducer (TMG) and one uninduced, were done.
    3. the phenotype of the merodiploid gradually switched from constitutive to induced over the course of a 2h period.
    4. this proved that "…the i gene in its active form controls the synthesis of a product which, when present in the cytoplasm, prevents the synthesis of beta-galactosidase and galactoside-permease, unless inducer is added externally…".

E.  The operator (Jacob and Monod, 1960)

Genotype 

phenotype 

i+o+z+

inducible 

i-o+z-(y-)/i+ocz+(y+) 

constitutive (for LacZ and LacY); therefore, oc is dominant and pleiotropic 

i+o+z-/i+ocz+ 

constitutive 

i+o+z+/i+ocz- 

inducible ; therefore, cis acting

i+o+z-y+/i+ocz+y-

constitutive for LacZ, inducible for LacY

1.          characterized another type of constitutive mutation, called oc.

2.        "The constitutive mutation oc is thus pleiotropic and dominant, and its effect is only manifested in the cis position."

3.        P.S. The operator was actually discovered before the promoter.

F. the Operon Concept

Positive regulation of the lac operon

A. back to diauxie

  1. What dictates the pattern in which glucose is used first, lactose is used only after glucose is gone?
  2. What is the state of the lac operon in the various phases of the diauxic growth curve?

B. catabolite repression is the term for the phenomenon that underlies the preference of bacteria for glucose (or any particular growth substrate).

C. cAMP and adenylate cyclase

D. CRP (the cAMP receptor protein)

  1. CRP is inactive by itself
  2. CRP-cAMP is able to bind near the lac promoter
  3. converts it from a weak to a strong promoter

E. the lac promoter is not strong by itself

  1. 5' TATGTT 3' is not a good match to consensus
  2. however, when CRP-cAMP binds to it, it becomes stronger.
  3. the lacUV5 promoter contains a double mutation that converts it from 5'TATGTT 3' to 5' TATAAT 3'
    1. is this promoter still subject to negative regulation?
    2. is it still subject to positive regulation?
    3. email your answer for zillions of bonus points

Summary of regulation of lac operon

A. Return to diauxie

  1. during first exponential phase, lac repressor is inactive, but so is CRP, so transcription of lac operon is minimal
  2. during middle plateau, cAMP builds up, and activates CRP.  The active complex binds and stimulates transcription of lacZYA.  Transcription is maximal, because both activation (CRP-cAMP) and derepression (lacI de-activation) are at work.

B. catabolite repression operates on several metabolic operons (maltose, arabinose, galactose, etc.), not just lac.  It's a general effect of glucose, which is the mother of all growth substrates for E. coli.

C. try this one: what is the state of the lac operon in cells growing on maltose only?   On maltose plus lactose?

Attenuation at the trp operon

a.      Different modes of regulation: catabolic vs. anabolic

1.        "Classical" negative regulation

2.        like lac, involves a repressor

3.        unlike lac, repressor is inactive as default state, becomes active in presence of the co-repressor tryptophan.

4.        genetic example of feedback inhibition

b.     attenuation

1.        this is a mechanism to achieve fine control over trp operon expression

1.        is a example of the tight coupling of transcription and translation in bacteria.

2.        involves a stretch of DNA called the attenuator region

3.        region contains transcriptional and translational signals

4.        translation signal is a short open reading frame that features two back-to-back trp codons (UGG UGG).

2.        think what will happen to translation when tryptophan is scarce in the cytoplasm.

1.        transcriptional signal consists of two possible stem loop structures: one is a terminator, and the other is an "antiterminator".

2.        if tryptophan is abundant, translation occurs without pause, and this favors the formation of the terminator.  Transcription of downstream structural genes (trpEDCBA) doesn't happen.

3.        if tryptophan is scarce, translation of the leader pauses at UGG UGG, and this favors the formation of the antiterminator. Transcription of trpEDCBA occurs..