Transcript PPT

REVIEW SESSION
Wednesday, September 15
5:30 PM
SHANTZ 242 E
Gene Regulation
Gene Regulation
Gene expression can be turned on, turned
off, turned up or turned down!
For example, as test time approaches, some of you may note
that stomach acid production increases dramatically….
due to regulation of the genes that control synthesis of
HCl by cells within the gastric pits of the stomach lining.
Gene Regulation in Prokaryotes
Prokaryotes may turn genes on and off depending on
metabolic demands and requirements for
respective gene products.
NOTE: For prokaryotes, “turning on/off” refers
almost exclusively to stimulating or repressing
transcription
Gene Regulation in Prokaryotes
Inducible/Repressible gene products: those
produced only when specific chemical substrates
are present/absent.
Constitutive gene products: those produced
continuously, regardless of chemical substrates
present.
Gene Regulation in Prokaryotes
Regulation may be
Negative: gene expression occurs unless it is shut off
by a regulator molecule
or
Positive: gene expression only occurs when a
regulator mole turns it on
Operons
In prokaryotes, genes that code for enzymes all
related to a single metabolic process tend to be
organized into clusters within the genome, called
operons.
An operon is usually controlled by a single
regulatory unit.
Regulatory Elements
cis-acting element: The regulatory region of the
DNA that binds the molecules that influences
expression of the genes in the operon. It is almost
always upstream (5’) to the genes in the operon.
Trans-acting element: The molecule(s) that
interact with the cis-element and influence
expression of the genes in the operon.
The lac operon
The lac operon contains the genes that must be
expressed if the bacteria is to use the disaccharide
lactose as the primary energy source.
To be used as an energy source, lactose must be
cleaved into glucose and galactose. The glucose is
then available for metabolism (glycolysis).
Note: glucose is the preferred energy substrate.
Negative Control
The genes in the lac operon are normally turned off,
and only expressed when a repressor molecule is
removed from the regulatory region.
This repressor is removed only in the presence of
lactose
The lac Operon
Repressor
gene
LacI
Regulatory
Region
P
O
P=Promoter
O=Operator
Structural Genes
lacZ
lacY
lacA
Structural Genes
Structural genes are those that encode for the
enzymes that do the metabolic work.
LacZ: b-galactosidase, cleaves lactose into glucose
and galactose
LacY: Permease, promotes entry of lactose into cell
LacA: Transacetylase, thought to reduce toxicity of
byproducts of lactose metabolism
Structural Genes
In prokaryotes, all the structural genes within an
operon are usually transcribed as a single mRNA,
then the genes are independently translated by
ribosomes.
LacI—The Repressor
LacI is the regulatory molecule.
When there is no lactose present in the cell,LacI
binds to the Operator element and blocks binding
of RNA polymerase to the Promoter element.
X
LacI
P
O
lacZ
lacY
lacA
LacI—The Repressor
When lactose IS present, the genes to metabolize
lactose must be expressed.
Lactose itself causes LacI to dissociate from the
operator, which frees up the promoter region,
allowing RNA polymerase to bind, and
transcription begins.
Lactose is the inducer molecule for the lac
operon.
Induction of the lac operon
Lactose
Binding of lactose causes a
change in the shape of LacI
LacI
P
O
lacZ
lacY
lacA
Induction of the lac operon
LacI
P
O
lacZ
lacY
lacA
LacI
P
O
lacZ
lacY
lacA
What happens if you mutate LacI?
LacI encodes the lac repressor, which keeps the
operon shut off in the absence of lactose.
What happens if you mutate LacI?
Inactivation of LacI would be called a constitutive
mutation, because the genes of the lac operon
would be on all the time even if there is no lactose
present (removed repression).
Positive Control of the lac Operon
A further increase in transcription of the lac operon
occurs if a molecule called catabolite-activating
protein (CAP) also binds the promoter region.
LacI
P
O
lacZ
lacY
lacA
CAP facilitates the binding of RNA polymerase,
and therefore increases transcription
Positive Control
Remember, glucose is the preferred substrate.
CAP exists in the state that will bind the promoter
ONLY when glucose is absent.
LacI
P
O
lacZ
lacY
lacA
This is the form CAP takes when there is no glucose
Positive Control
When glucose is present, CAP exists in a state that
will NOT bind the promoter of the lac operon.
LacI
P
O
lacZ
lacY
lacA
X
This is the shape CAP takes when glucose is
present. It cannot bind the promoter in this shape
X
Regulation of the lac Operon
So, transcription is regulated as follows:
Off when lactose is absent (repressed)
Active when lactose is present as well as glucose
(de-repressed)
Really active when lactose is present but glucose is
absent (activated)
Gene Regulation in Eukaryotes
Differences between Prokaryotes and
Eukaryotes
1. DNA is a lot more complicated in eukaryotes—
there’s a lot more of it and it’s complexed with
proteins to form chromatin
2. Genetic information is carried on multiple
chromosomes
3. Transcription and translation are physically
separated
Differences between Prokaryotes and
Eukaryotes (cont.)
4. Eukaryotic mRNA is processed prior to translation
5. Eukaryotic mRNA is much more stable (not as
easily degraded)
Gene expression can be controlled at the level of
translation!
6. Different cell types express different genes
Chromatin Remodeling
Chemical alteration of the histone proteins of
chromatin facilitates or inhibits access of RNA
polymerases to DNA promoters.
Recruitment of Co-activators
Remember enhancer elements? These are
binding sites for molecules that influence
formation of the RNA polymerase initiation
complex.
Enhancer elements may have DNA sequences for
both positive and negative regulators of
transcription.
Enhancers
The presence or absence of regulators is determined by the
cell’s environment, metabolic state, developmental state
and/or the presence or absence of signal molecules.
The net effect of all the information available, summed up
by the regulators present, dictates the transcription
efficiency of RNA polymerase from a given promoter.
DNA Methylation
Chemical modification of DNA by adding or
removing methyl (-CH3) groups from the DNA
bases, usually cytosine.
The presence of the methyl group alters the shape of
DNA, which influences the binding of proteins to
the methylated DNA.
DNA Methylation
Typically, increased methylation decreases
transcription efficiency.
In mammalian females, one X chromosome is
inactivated (only one of the X chromosomes is
used to drive transcription). The inactivated X
chromosome has much more methylation than the
active chromosome.
Post-Transcriptional Regulation
Alternative Splicing:
Exon 1
1.
Exon 2
Exon 1
2.
Exon 3
Exon 2
Exon 1
Exon 3
Exon 2
Exon 4
Exon 4
Exon 4
Exon 5
Exon 5
Exon 5
Post-Transcriptional Control
RNA Stability
1. Stability sequences
2. Instability sequences
3. Translation efficiency—increased translation
increases stability