Transcript Presentation

What makes cells from the same individual look
different?
Stem Cells
Red Blood Cells
Liver Cells
Cartilage Cells
DNA sequence in each cell is the same, but different cell types
have different “GENE EXPRESSION PATTERNS”
• When a gene is “on” and its
protein or RNA product is being
made, scientists say that the gene is
being EXPRESSED.
• The on and off states of all of a
cell’s genes is known as a GENE
EXPRESSION PROFILE.
• Each cell type has a unique gene
expression profile.
Insulin
Muscle Cell
Pancreatic Cell
DNA?
Protein?
X
Gene Expression and Regulation
KEY CONCEPT
Gene expression is carefully regulated in both
prokaryotic and eukaryotic cells.
http://www.cpalms.org/Public/PreviewResourcePV/Preview/145675
Gene Regulation in Prokaryotes
• Bacteria adapt to changes in their
surroundings by using regulatory proteins to
turn groups of genes on and off in response to
various environmental signals.
• The DNA of Escherichia coli is sufficient to
encode about 4000 proteins, but only a
fraction of these are made at any one time. E.
coli regulates the expression of many of its
genes according to the food sources that are
available to it.
Prokaryotic cells turn genes on and off
by controlling transcription.
• An operon is a cluster of bacterial genes along with an
adjacent promoter that controls the transcription of those
genes.
• An operon includes a promoter, an operator, and one or
more structural genes that code for all the proteins needed
to do a job.
• A promotor is a DNA segment that allows a gene to be
transcribed.
• An operator is a part of DNA that turns a gene “on” or
”off.”
The Lac Operon
– Operons are most common in prokaryotes.
– The lac operon was one of the first examples of
gene regulation to be discovered.
– The lac operon has three genes that code for
enzymes that break down lactose.
• The lac operon acts like a switch.
– The lac operon is “off” when lactose is not
present.
– The lac operon is “on” when lactose is present.
• When the genes in an operon are transcribed,
a single mRNA is produced for all the genes in
that operon.
• This mRNA is said to be polycistronic because
it carries the information for more than one
type of protein.
• The operator is a short region of DNA that lies
partially within the promoter and that
interacts with a regulatory protein that
controls the transcription of the operon.
• Here's an analogy.
• A promoter is like a doorknob, in that the promoters
of many operons are similar.
• An operator is like the keyhole in a doorknob, in that
each door is locked by only a specific key, which in
this analogy is a specific regulatory protein.
The regulatory gene lacI produces an mRNA that
produces a Lac repressor protein, which can
bind to the operator of the lac operon.
• Regulatory genes are not necessarily close to the
operons they affect.
• The general term for the product of a regulatory gene
is a regulatory protein.
• The Lac regulatory protein is called a repressor
because it keeps RNA polymerase from transcribing
the structural genes.
• Thus the Lac repressor stops transcription of the lac
operon
• In the absence of lactose, the Lac repressor
binds to the operator and keeps RNA
polymerase from transcribing the lac genes.
– It would be energetically wasteful for E. coli if the
lac genes were expressed when lactose was not
present.
• The effect of the Lac repressor on the lac genes
is referred to as negative regulation.
– When lactose is present, the lac genes are expressed
because allolactose binds to the Lac repressor
protein and keeps it from binding to the lac
operator.
– Allolactose is an isomer of lactose. Small amounts of
allolactose are formed when lactose enters E. coli.
– Allolactose binds to an site on the repressor protein
causing it to change shape. As a result of this change,
the repressor can no longer bind to the operator
region and falls off. RNA polymerase can then bind
to the promoter and transcribe the lac genes.
• Allolactose is called an inducer because it
turns on, or induces the expression of, the lac
genes.
• The presence of lactose (and thus allolactose) determines
whether or not the Lac repressor is bound to the operator.
– Allolactose binds to an site on the repressor protein
causing it to change shape.
– As a result of this change, the repressor can no longer
bind to the operator region and falls off.
– RNA polymerase can then bind to the promoter and
transcribe the lac genes.
– When the enzymes encoded by the lac operon are
produced, they break down lactose and allolactose,
eventually releasing the repressor to stop additional
synthesis of lac mRNA.
Messenger RNA breaks down after a
relatively short amount of time.
• Whenever glucose is present, E. coli metabolizes it before using
alternative energy source such as lactose.
• Glucose is the preferred and most frequently available energy
source for E. coli. The enzymes to metabolize glucose are made
constantly by E. coli.
• When both glucose and lactose are available, the genes for lactose
metabolism are transcribed at low levels.
• Only when the supply of glucose has been exhausted does does
RNA polymerase start to transcribe the lac genes efficiently, which
allows E. coli to metabolize lactose.
• When both glucose and lactose are present, the genes for lactose
metabolism are transcribed to a small extent
Eukaryotes regulate gene expression at
many points.
• Different sets of genes are expressed in
different types of cells.
• Transcription is controlled by regulatory DNA
sequences and protein transcription factors.
• Transcription is controlled by regulatory DNA sequences
and protein transcription factors.
– Most eukaryotes have a TATA box promoter.
– This is the sequence RNA polymerase recognizes
and binds to: how it “knows” where a gene starts
– Enhancers and silencers speed up or slow down
the rate of transcription.
– Each gene has a unique combination of
regulatory sequences.
Eukaryotic mRNA Processing
• RNA processing is also an important part of gene regulation
in eukaryotes.
• mRNA processing includes three major steps.
• mRNA processing includes three major steps.
– Introns are removed and exons are spliced together.
– A methyl cap is added.
– A poly-A tail is added.
• Unlike prokaryotes which have one RNA
polymerase that makes all classes of RNA
molecules, eukaryotic cells have three types of
RNA polymerase (called RNA pol I, RNA pol II,
and RNA pol III), and each type of RNA is made
by its own polymerase:
– RNA polymerase I makes ribosomal RNA (rRNA)
– RNA polymerase II makes messenger RNA (mRNA)
– RNA polymerase III makes transfer RNA (tRNA)
• RNAs are made in the nucleus of a eukaryotic
cell, but function in protein synthesis in the
cytoplasm.
• Unlike prokaryotic mRNAs, eukaryotic mRNAs
undergo extensive modifications after synthesis
by RNA polymerase II.
• These changes include capping, polyadenylation,
and splicing.
Adding the Methyl Cap
– Modification of the 5'-ends of eukaryotic mRNAs is
called capping.
– The cap consists of a methylated GTP.
– Capping occurs very early during the synthesis of
eukaryotic mRNAs, even before mRNA molecules are
finished being made by RNA polymerase II.
– Capped mRNAs are very efficiently translated by
ribosomes to make proteins.
– In fact, some viruses, such as poliovirus, prevent
capped cellular mRNAs from being translated into
proteins. This enables poliovirus to take over the
protein synthesizing machinery in the infected cell to
make new viruses.
Polyadenylation (Poly-A tail)
• Modification of the 3'-ends of eukaryotic mRNAs
is called polyadenylation.
• Polyadenylation is the addition of several
hundred A nucleotides to the 3' ends of mRNAs.
• This string of A’s makes the poly-A tail.
Splicing
– Eukaryotic genes are often interrupted by sequences
that do not appear in the final RNA.
– The intervening sequences that are removed are
called introns.
– The process by which introns are removed is referred
to as splicing.
– The sequences remaining after the splicing are called
exons.
– Although most higher eukaryotic genes have introns,
some do not.
– Higher eukaryotes tend to have a larger percentage of
their genes containing introns than lower eukaryotes,
and the introns tend to be larger as well.
– The pattern of intron size and usage roughly follows
the evolutionary tree, but this is only a general
tendency.
– The human titin gene has the largest number of exons
(178), the longest single exon (17,106 nucleotides)
and the longest coding sequence (80,781 nucleotides
= 26,927 amino acids).
– The longest primary transcript, however, is produced
by the dystrophin gene (2.4 million nucleotides).
Usefulness of splicing
• Splicing has evolutionary implications.
– Exons often coincide with protein "domains”.
– Domains are parts of the protein with a specific
function.
– Exons can be readily "exchanged" between different
genes by recombination.
– This means that new types of proteins can be formed
relatively easily.
• Splicing also allows a cell to "swap" exons during
gene expression.
– For example, during development, some genes are
spliced one way, and then spliced a different way
later.
– Changing the way a mRNA is spliced changes the
amino acid sequence in the protein made from it, so
cells can in this way "modify" the sequence, and
function, of a protein.
– Splicing is yet another mechanism for regulating
whether or not a specific version of a protein is
made, how much of it is made, and when it is made.
• One very good example of exon shuffling can be
seen in the tropomyosin gene.
• Tropomyosin is a protein involved in muscle-like
contraction in cells.
• It is present in many different types of cells of the
body.
• The tropomyosin mRNAs in different types of muscle
cells are slightly different from each other, but all
come from the same gene.
Discovery of RNA Interference or RNAi
• During the research of Andrew Fire and Craig
Mello on gene expression in the worm C.
elegans, they found that injecting mRNA that
encodes for muscle protein production elicited
no responses from the worms.
– Bear in mind that the genetic code in the mRNA is
considered as the sense sequence.
– They also tried to inject antisense RNA into the
worms which can pair with the sense sequence
mRNA but it also elicited no responses from the
worms.
– Finally, when they tried to inject both the sense and
the antisense RNA together, they noted twitching
movements from the worms.
• These results surprised them since they know
that the same kinds of movements were noted
from worms whose genes encoding for muscle
protein were dysfunctional.
• To explain the results that they got, Fire and
Mello hypothesized that the double-stranded
RNA molecule formed by the binding of the
sense and antisense RNA silences the gene
carrying exactly the same code as the RNA
molecule.
• To test their hypothesis, they injected doublestranded RNA that codes for specific proteins.
• In all their experiments, they found that the
genes carrying exactly the same code as the RNA
they injected were silenced.
• Their discovery on RNA interference is
noteworthy for two reasons.
– First, with RNAi, researchers can specifically
knockdown the production of any protein in a cell.
– Second, initially scientists thought that a portion of
the DNA called introns were just junk DNA and they
serve very little purpose, buy now they know that
much of these introns code for RNAi elements.
siRNA
• Small interfering RNA (siRNA) are small pieces of
double-stranded (ds) RNA that can be used to
"interfere" with the translation of proteins by
binding to and promoting the breakdown of
messenger RNA (mRNA) at specific sequences. In
doing so, they prevent the production of specific
proteins.
• The process is called RNA interference (RNAi), and may
also be referred to as siRNA silencing or siRNA
knockdown.
– siRNA often comes from vectors, like viruses, and have
been found to play a role in antiviral defense,
degradation of over-produced mRNA or mRNA for
which translation has been aborted, and preventing
disruption of genomic DNA by transposons.
– The siRNA then "seeks out" an appropriate target
mRNA, where the siRNA then causes the mRNA to be
broken down.
• Many diseases can potentially be treated by
inhibiting gene expression.
– Therefore, the design of synthetic siRNA for
therapeutic uses has become a popular objective of
many biopharmaceutical companies.