BIOL SCI 215 Lecture Notes - Lecture 9: Plasmid, Lac Operon, Beta-Galactosidase
28 Jun 2018
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Gene regulation
“Some are always on” - constitutive [expressed]
Some can be turned on or off - regulatable
Gives organism ability to respond to environment or generate complexity
Two possible ways for regulation:
oPositive regulation: Activator is facilitating transcription by binding to DNA and
in absence, transcription does not occur
oNegative regulation: DNA binding factor - DNA repressor binding prevents
transcription
E.coli - single cell bacteria, consumes glucose and lactose [two 6
member rings]
Plasma membrane is hydrophobic and thus galactoside
permease helps lactose enter the cell
Key step to metabolize this lactose is beta-
galactosidase in which it breaks down lactose to
glucose and galactose
Permeates protein only when lactose is present
1. Before exposure to lactose, they do not express enzyme beta-
gal or permease
2. Add lactose and the beta is expressed and encoded from the
lacZ gene
Fool cell that there is lactose by having IPTG compound which increases the beta-gal
abundance and it is not consumed. Since IPTG can’t be degraded, beta-gal remains
expressed
Mechanism of translation: Prok have shine delgano which binds to 16S, allowing
them to translate ORF which are internal so they can have multiple sequences on the
mRNA
Polycistronic gene structure: Bacteria houses genes that have the same sequences
Lac operon: Beta gal is the enzyme that takes lactose and breaks it down
DNA Binding factor called Lac-I has its own promoter and terminator - constituitively
expressed so it makes an mRNA that forms a protein that has a structure which
allows for recognition of gene sequences
LacI binds to LacO - prevents polymerase from moving forward and transcribing the
genes
When lactose is present, LacI is still present and transcribed. But because lactose
enters the cell and changes the lacI shape, thus preventing it from binding to lac o,
and thus the whole sequence lacZ, lacY and LacA ORFs will be read and
transcribed. Each lac have their shine dalgarno start and stop codons.
LacI is needed for operon function and is known as the repressor because it
represses expression of LacZYA. Also its prime function is regulating expression of
genes
oPromoter can be located anywhere in the genome
LacI repressor encodes a protein that binds the lacO DNA
LacI binds to LacO (the operator) by recognizing specific bases
What happens if the different parts were missing?
Wildtype situation and 2 mutant phenotypes [Beta gal expression is always on/ never on]
LacI
LacI cant bind and doesnt matter if lactose is present and will never get LacZ
expression
LacO
LacI cant bind and thus we will always have LacZ expressed in the individuals
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Document Summary
Some can be turned on or off - regulatable. Gives organism ability to respond to environment or generate complexity. Two possible ways for regulation: positive regulation: activator is facilitating transcription by binding to dna and in absence, transcription does not occur, negative regulation: dna binding factor - dna repressor binding prevents transcription. E. coli - single cell bacteria, consumes glucose and lactose [two 6 member rings] Plasma membrane is hydrophobic and thus galactoside permease helps lactose enter the cell. Key step to metabolize this lactose is beta- galactosidase in which it breaks down lactose to glucose and galactose. Permeates protein only when lactose is present. Before exposure to lactose, they do not express enzyme beta- Add lactose and the beta is expressed and encoded from the. Fool cell that there is lactose by having iptg compound which increases the beta-gal abundance and it is not consumed. Since iptg can"t be degraded, beta-gal remains expressed.
