Transcript Bio1A Unit 2-7 Gene Expression Pt 1 Notes File

Mutation: by function
• Mutants are almost always “loss of function”  whatever you mutated doesn’t work. There are
far more ways for things to go wrong than right. The alternative is a “gain of function” mutation.
• Think of mutations in terms of an enzyme.
substrate
enzyme
Product
Ex: Pigment = Red
Mutant
substrate
No Product
Not red
• mutations are just in DNA sequence. There is more non-coding DNA than coding in the
(human) genome.
• We will consider regulatory elements which are genes AND promoter elements
Control of Gene expression
• Gene is “expressed” when:
– It is transcribed
– RNA is translated
– Protein is active
• organisms respond to environment by controlling the
expression of their genes
• Multi-cellular organisms express different genes in
different cell types
Lactose metabolism in bacteria
• Bacteria can digest lactose - requires proteins / enzymes. i.e. – β-galactosidase
• Bacteria will not produce lactose metabolic enzyme unless needed  Lactose present
• If lactose is absent enzyme are not made in order to conserve energy  No β-gal
• Bacteria prefer Glucose (fewer enzymatic steps = fewer enzymes = more efficient)
• When glucose is present lactose enzymes not make
Efficient = growth advantage
Inefficient bacteria don’t die, they are just become minority (subpopulation)
Operons – Bacteria, not Eukaryotes
• entire stretch of DNA that includes the operator, the promoter, and the genes that they control
• cluster of (related) genes can be under coordinated control by a single on-off “switch” (Operator)
• operator usually positioned within the promoter
Promoter
Operator
Multiple SD - start/stops
Lac Repressor
• regulatory gene / separate protein under another promoter distinct from the operon
• repressor binds to operator & prevents transcription by blocking RNA polymerase
Lac Operon is inducible
• By itself, the lac repressor is active and switches the lac operon off
• A molecule called an inducer inactivates the repressor to turn the lac operon on
• The inducer, lactose, inactivates lac repressor to turn lac operon on
Experimental Analysis of Gene Regulation
Tools Specific to Lactose Regulation
Substrate analogs - A compound that “looks” like the native (original) substrate
IPTG
Looks like allolactose
 Inducer of LacI
But not degraded
X-gal
Indicator
Looks like lactose (Not an inducer)
Recognized by β-gal
Used to detect:
the expression
(presence) of β-gal
= if lac operon is on
Produces blue
precipitate
Lac Operon Discovery
Genetics: find genes & other regulatory elements
Mutagenesis Experiment
X-gal
IPTG
X-gal
NO IPTG
NO inducer
LacI Repressor binds
 Block RNA Pol
 No transcription
 No β-gal
 doesn’t digest X-gal
Mutagenesis
X-gal
NO IPTG
+ inducer
LacI Repressor inactivated
Transcription
β-gal made
digests X-gal  Blue
Lac I mutant (loss of function)
NO inducer
LacI Repressor binds
 Block RNA Pol = No transcrip.
 No β-gal
 doesn’t digest X-gal
Transcription
β-gal made
digests X-gal  Blue
Generically: In a mutagenesis experiment
• Mutations in a negative regulator will result in a constitutive mutant (always on, even when it
would normally be off)
• Mutations in a positive regulator will result in an uninducible mutant (always off, even when it
would normally be on)
Glucose regulates lac operon
Glucose
• Best energy source
 directly enters glycolysis
• All other compounds require energy to modify
• E. Coli will not induce lac operon if glucose is
present. Even when lactose inducer is present
• When Glucose is present cAMP is not produced
Glucose  no Camp
 No CAP binding
 No transcription  White
No Glucose  cAMP
 cAMP bind CAP
 CAP binds CAP binding site
 RNA polymerase attracted
 Gene expression turned on
= β-gal made
 β-gal digests x-gal
 Blue
Repressible and Inducible Operons: Two Types of Negative Gene Regulation
• An inducible operon is one that is usually off; an inducer inactivates the repressor (or activates
and activator) and turns on transcription
• A repressible operon is one that is usually on; corepressor gives binding of a repressor to the
operator shuts off transcription
• The lac operon is an inducible operon
• The trp operon is a repressible operon
• The repressor can be in an active or inactive form, depending on the presence of other
molecules
• A corepressor is a molecule that cooperates with a repressor protein to switch an operon off
• Operator & repressor, activator/enhancer, inducer, corepressor
Terminology
Repressors:
• Protein - ↓ gene expression
• binds to upstream elements to block RNA polymerase / transcription
Activtators: (a.k.a. – enhancer)
• Protein -↑ gene expression
• binds to upstream elements to aid RNA polymerase binding / transcription
Inducers, Corepressor, Coactivators
(prok. vs euk.) – small molecules
Inducers:
• non-protein, small molecules - ↑ gene expression (either by binding and removing repressors or
binding activators to cause them to bind their activator binding site)
Corepressors:
• prokaryotes: non-protein, small molecules - ↓ gene expression (either by removing activators or
causing repressor to bind)
• Eukaryotes: protein that bind repressors to ↓ gene expression but don’t bind DNA themselves
Coactivtators: (only eukaryotes)
• Protein that binds activator to ↑ gene expression , but doesn’t bind DNA itself (only eukaryotes)
•
No analogous term in prokaryotes and so the terms corepressor and coactivator are blurred for
prokaryotes and eukaryotes
Eukaryotic Genes have multiple enhancers for transcription factors
vs Lac operon only has room next to promoter
E1
E2
E3
E4
Far away from promoter
TATA
While no operon system in eukaryotes,
coordinate regulation is accomplished by
genes having the same enhancer elements
Promoters typically have negligible basal activity.Can be regulated by multiple enhancers for complex
decisions that vary due to conditions such as:
• Cell types
• Specific time in development
• Specific physiological conditions