Lecture 10

Gene Regulation I: Promoters and Control Circuits

SPs: Figs 12-27, 28, 29, 32, 40, 44

Vocabulary:operon/regulatory gene/repressor/inducer/ co-repressor/derepression/positive vs. negative control/ cis vs. trans acting factors/deletion mutant/enhancer

Fig 12-28

Operon Structure

Lactose= disaccharide of galactose + glucose

Lactose (inducer)

β-galactosidase expressed

lactose glucose + galactose

Lactose metabolism in E. coli

Inducible Operon: The Lac operon

If Lac present, repressor inactivated

Operon induced, mRNA transcribed

Fig 12-29

This is ‘Induction’

Translation of mRNA yields 3 enzymes that convert:

Lactose Glucose + Galactose

Repressor now able to bind to the operator

Repression occurs: transcription blocked

What happens when lactose supply is reduced?

Binding of lactose to repressor is transient, so as [lactose] falls, repressor becomes active

Repressible Operon: TRP Operon

•  If TRP present, repression occurs

•  TRP acts as corepressor

Fig 12-29

Used to make the amino acid tryptophan

Default is ON, unless TRP is present

Repressed state: no TRP production

Derepression (reactivation) occurs

Thus no co-repressor present & repressor no longer functions

As TRP used, [TRP] falls

mRNA processed and translation yields 5 enzymes that convert precursors to tryptophan

Positive vs. negative control: depends on the active form of the trans-acting factor (e.g. repressor), and its effect upon binding to its target cis-acting sequence.

Cis vs. Trans: ‘Cis-acting promoter sequence to which a trans-acting transcription factor binds

promoter

Trans: (trans-acting/ e.g. a transcription factor)

Cis: “on the same strand”; e.g. DNA sequence that serves as a binding site for a TF.

Coding region

Bacterial Biochemical Logic for LAC=GLU + GAL

1.  If glucose is available, why expend energy to make enzymes to catabolize lactose?

2. If lactose is absent, why expend energy to make enzymes to catabolize it?

Both positive and negative control involved.

Positive control: if glucose level is low, cAMP level is high; cAMP binds to CRP, and the complex activates the lac operon.

Allolactose: an isomer of lactose, is the actual inducer

Four situations: sugar availability and positive/negative control

Brooker Fig 12.11

There are four LAC operon and two TRP operon animation links available on Blackboard- have a look!

Fig 12-32

Post-translational control also may exist

Overview of levels of control of gene expression

Transcription level control:identifying promoter motifs

Components involved:

1. Recombinant DNA methods to alter sequences

2. Method of transforming cells of interest

•  ‘Reductionist’ approach: remove/alter sequence

3. Method of evaluating transcription

•  Hybridization using radioactive probe

•  Reporter gene technology

Coding region promoter TATA box

Step 1: Altering cloned DNA

Recombinant DNA methods permit almost any change to your favorite gene (YFG)

Deletions:

YFG TATA box ? ? ?

YFG

YFG

Site-directed mutagenesis is also possible:

Example: 5’ -GACCATGCT- changed to: 5’ -GACTATGCT-

Step 2: Transform target cells with the altered DNA

•  Typically this was the original ‘host’

•  Many methods available to transform cells

•  Problem: how to analyze? Endogenous (normal gene) already present, interferes with analysis of the altered DNA

Step 3: Analysis of expression of the ‘transgene’

•  ‘Transgenic’ organism produced.

•  Solution? Use a ‘reporter gene’

e.g: β-galactosidase or Green fluorescent protein (GFP)

Reporter genes: examples

FIL::GFP shows expression on the dorsal face of floral organs: symmetry

MYO::β-galactosidase in mouse embryo: muscle development

Histochemical staining/fluorescence reveal expression pattern

Fig 12-33

6. Deletion in this region = higher levels of product A negative element has been removed by deletion.

Analysis of deletion mutants defines cis-acting regulatory sequences

Fig 12-40

Many promoters contain binding sites for a number of proteins that can influence transcription.

Fig 12-43

Multiple binding sites: provide fine control over expression and the ability to respond to multiple signals.

• DNA elements often located far away from the gene they control.

Enhancers:

• Orientation independent. Why?

1

1. Chromatin modifications

2

2. Interactions with general transcription machinery

Fig 12-44

Protein factors that influence transcription

Transcription factors:

Positive and/or negative roles: e.g. may act to activate one gene and repress another

Next class: Transcription factors and microarray (gene chip) technology