BCMB2901 Lecture Notes - Lecture 7: Lac Operon, Operon, Ampicillin
15 May 2018
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Lecture 7: Prokaryotic transcription (Transcriptional Regulation I)
Gene expression responds to environmental conditions
• Some regulatory proteins present at 5-10 copies/ cell, some enzymes for glycolysis present at
100,000 copies/cell
• Genes whose products are presently unneeded or at acceptable levels are turned off
• Mechanisms to achieve proper mix of gene expression are varied
Lactose metabolism in E. coli
• Enzyme β-galactosidase only expressed when lactose present in the medium
o Enzyme said to be expressed in inducible fashion with lactose as inducer
• Identified cis-acting elements (operator, promoter) & trans-acting factors
Lactose hydrolysis
• Enzyme encoded by lacZ gene
• Glucose & galactose products
• Enzyme cleaves broad range of β-galactosides (including synthetic analogs eg. X-gal)
E. coli lac operon
• LacI has constitutive promoter & is expressed separately from lac operon
o Encodes lac repressor
o Low level of expression
• The E. coli lac operon is an inducible set of genes
• These genes are responsible for the breakdown of lactose à sugars for cellular metabolism
Cloning: Blue-white selection
Resistance to ampicillin
Functional β-
galactosidase?
X-gal color
Empty plasmid
Yes
Yes
Colorless
Recombinant plasmid
with DNA insert
Yes
No (disrupted lacZ gene)
Blue (ppt)
Lac Operon expression (gene regulation)
• RNA polymerase: builder & needs a promoter
• Operator is next to promoter & is where repressor is binded (blocks RNA polymerase so no RNA
made)
• Lac Operon expression: encodes polycistronic mRNA giving rise to 3 diff enzymes
o 3 enzymes (LacZ, lacY, lacA) help break down lactose
o Enzymes encoded are β-galactosidase (lactose à glucose + galactose) , lactose permease
(membrane protein that transports lactose into cells), transacetylase
• lacI codes of production of repressor (has its own promoter)
• if there is no lactose, repressor binds to operator, blocks RNA polymerase, no RNA and thus protein
produced
• if there IS lactose, it binds to the repressor, change the repressor conformation & so it cannot bind
to operator, RNA polymerase can go through and thus RNA is produced (expressing operon)
o the mRNA will make enzymes to break down the lactose sugar
The promoter & operator overlap
Gratuitous Inducers
A molecule structurally similar to another molecule that
induces transcription for a specific product, & can initiate
transcription for that product when official inducer is absent
• Lactose: normal inducer, but β-galactosides also work
• Isopropylthiogalactoside (IPTG) also acts as an
inducer but is not metabolized
o Level remain constant
o Shows induction does not involve interaction
with the actual enzyme being synthesized
The lac promoter is quite weak
Controlling region
Document Summary
Gene expression responds to environmental conditions: some regulatory proteins present at 5-10 copies/ cell, some enzymes for glycolysis present at. 100,000 copies/cell: genes whose products are presently unneeded or at acceptable levels are turned off, mechanisms to achieve proper mix of gene expression are varied. Lactose metabolism in e. coli: enzyme -galactosidase only expressed when lactose present in the medium, enzyme said to be expressed in inducible fashion with lactose as inducer. Identified cis-acting elements (operator, promoter) & trans-acting factors. A molecule structurally similar to another molecule that induces transcription for a specific product, & can initiate transcription for that product when official inducer is absent: lactose: normal inducer, but -galactosides also work. Isopropylthiogalactoside (iptg) also acts as an inducer but is not metabolized: level remain constant, shows induction does not involve interaction with the actual enzyme being synthesized. Cap needs camp to enable it to bind promoter (conformation is incorrect for binding)
