Lecture 10 Gene Regulation I: Promoters and Control Circuits SPs: - - PowerPoint PPT Presentation

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

• f 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

• n 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

• f 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