Download Chapter 17 Presentation Transcription Translation and Gene

Chapter 17
From Gene to Protein
Main Questions:
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The information content in DNA is the
specific sequence of nucleotides along the
DNA strands.
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How does this information determine the
organism’s appearance?
How is the information in the DNA sequence
translated by a cell into a specific trait?
The Bridge Between DNA and
Protein
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RNA is the single stranded compound that
carries the message from the DNA to the
ribosome for translation into protein.
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Recall, DNA = A,T,C,G; RNA= A,U,C,G
The order of these bases carries the code
for the protein which is constructed from
any or all of the 20 amino acids.
Transcription and Translation
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Going from gene to protein.
Transcription is the synthesis of mRNA
using DNA as the template, and is similar
to DNA synthesis.
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mRNA is the message (hence the “m”) from the
gene.
Translation is the process that occurs when
the mRNA reaches the ribosome and
protein synthesis occurs.
RNA
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RNA is used because it is a way to protect
the DNA from possible damage.
Many copies of RNA can be made from
one gene, thus, it allows many copies of a
protein to be made simultaneously.
Additionally, each RNA transcript can be
translated repeatedly--via polyribosome.
Recall the Main Difference
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Between prokaryotes and eukaryotes,
there is one main difference between
transcription and translation. The two
processes can occur simultaneously in
prokaryotes because they lack a nucleus.
In eukaryotes, the two processes occur at
different times. Transcription occurs in the
nucleus, translation occurs in the
cytoplasm.
The Genetic Code
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61 of the 64 codons
code for amino acids.
3 of the codons code
for stop codons and
signal an end to
translation.
AUG--start codon
Genetic Code
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The genetic code is said to be redundant.
More than one triplet codes for the same
amino acid.
One triplet only codes for one amino acid.
The reading frame is important because
any error in the reading frame codes for
gibberish.
Transcription
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The gene determines the sequence of
bases along the length of the mRNA
molecule.
One of the two regions of the DNA serves
as the template.
The DNA is read 3’-->5’ so the mRNA can
be synthesized 5’-->3’
Translation
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mRNA triplets are called codons.
Codons are written 5’-->3’
Codons are read 5’-->3’ along the mRNA
and the appropriate aa is incorporated into
the protein according to the codon on the
mRNA molecule.
As this is done, the protein begins to take
shape.
mRNA and RNA Polymerase
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mRNA is the “messenger” or vehicle that
carries the genetic information from the
DNA to the protein synthesizing machinery.
RNA polymerase pries apart the DNA and
joins RNA nucleotides together in the 5’-->3’
direction (adding, again, to the free 3’ end).
RNA polymerase is just like DNA
polymerase, but it doesn’t need a primer.
The Synthesis of mRNA
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RNA pol II encounters
a promoter on the DNA
near a transcriptional
unit and starts
synthesizing RNA.
When the RNA pol II
encounters a
terminator sequence,
transcription stops.
The Synthesis of mRNA
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RNA pol II encounters a promoter
on the DNA near a transcriptional
unit and starts synthesizing RNA.
When the RNA pol II encounters a
terminator sequence, transcription
stops.
Different Types of RNA
Polymerase
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Prokaryotes have one type of RNA
polymerase that synthesizes mRNA and
the other types of RNA as well.
Eukarytoes have 3 different types in their
nuclei (I, II, III). mRNA synthesis uses
RNA pol II.
Promoters
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Promoters are found on the DNA molecule
and initiate the transcription of the gene.
This is the site where transcription factors
and RNA polymerase attach.
Promoters
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Transcription is finished when the RNA
polymerase reaches the terminator.
The stretch of DNA that is transcribed is
known as the transcription unit.
Promoters serve as great examples of noncoding DNA that has a function.
Promoters
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Promoters are specific base sequences to
which specific transcription factors (proteins)
bind to initiate gene expression.
They usually extend a few dozen
nucleotides upstream from the transcription
start point.
Include a “TATA box” in eukaryotes.
Promoters are important for the binding of
the RNA polymerase.
The Initiation of Transcription
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Transcription factors
bind to the promoter
region enabling RNA pol
II to do so.
The RNA pol II binds
with additional
transcriptional factors
creating a transcription
initiation complex.
DNA unwinds and
transcription begins.
RNA pol II
Promoter Differences Between
Prokaryotes and Eukaryotes
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In prokaryotes, RNA polymerase
recognizes and binds to the promoter on
the DNA associated with sigma factor
proteins and immediately begins
synthesizing mRNA.
In eukaryotes, a group of proteins called
transcription factors are needed for the
binding of the RNA polymerase and the
initiation of transcription.
Promoter Differences Between
Prokaryotes and Eukaryotes
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Once the transcription factors
bind to the promoter, RNA pol
II binds and transcription can
then proceed.
The entire group of proteins in
the eukaryote are called the
transcription initiation
complex.
Transcription
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As the RNA pol II moves along the DNA, it
uncoils it, synthesizes the mRNA transcript
and peels away from the DNA allowing it to
recoil.
Numerous RNA polymerases can
transcribe the same DNA segment (protein)
at the same time.
This enables the cell to make large
amounts of protein in a short period of time.
Transcription
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An electron micrograph showing the transcription
of 2 genes.
Transcription Termination
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In prokaryotes transcription proceeds
through a DNA sequence that functions as
a termination signal causing the
disassembly of the transcription complex
and the polymerase to detach from the
DNA.
This release of the transcript makes it
immediately available for use as mRNA in
prokaryotes.
Transcription Termination
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In eukaryotes, when the RNA pol II reads a
certain signal sequence, it cleaves off the RNA
from the growing chain as RNA pol II continues
transcribing DNA.
The RNA pol II continues to read and transcribe
DNA and eventually falls off the DNA template
strand, (not fully understood).
The RNA produced now is still not ready for use.
RNA Modification
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The eukaryotic RNA
transcript now gets
modified before it
enters the cytoplasm.
The 5’ end of the transcript gets modified before
leaving the nucleus--a 5’ cap of nucleotides.
The 3’ end is also modified--numerous adenine
nucleotides--called a poly-A tail.
Important Functions of the 5’ Cap
and Poly-A Tail
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They facilitate export of the mature mRNA
from the nucleus.
They protect mRNA from degradation by
hydrolytic enzymes.
They assist in the attachment of the
ribosome to the 5’ end of the mRNA.
mRNA Modification
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The mRNA is further
processed after the ends
have been modified--RNA
splicing.
The initial transcript
(~8000 bp) is reduced (to
~1200 on average).
The large, non-encoding
regions of the DNA that
get transcribed are spliced
out.
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Introns--intervening regions
are removed.
Exons--expressed regions
are kept.
mRNA Modification
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Some untranslated regions of the exons
are saved because they have important
functions such as ribosome binding.
RNA splicing occurs via snRNP’s.
snRPs consist of RNA and protein and join
together to form a spliceosome which
interacts with the intron to clip it out and
join the exons together.
So, Why is RNA Splicing
Significant?
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In many genes,
different exons
encode separate
domains of the protein
product.
RNA Splicing
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The way the RNA is spliced determines which
proteins will be expressed.
The different sexes of some organisms splice RNA
differently and thus translate the genes into
proteins differently--contributing to differences seen
among sexes.
The alternative RNA splicing is one possible
reason humans can get by with relatively few
genes.
Translation
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Translation is when the cell interprets the
genetic message and builds the
polypeptide. tRNA acts as the interpreter.
tRNA transfers aa’s from the cytoplasm to
the ribosome where they are added to the
growing polypeptide.
All tRNA molecules are different.
tRNA Structure and Function
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tRNA, like mRNA, is made
in the nucleus and is used
over and over again.
tRNA binds an aa at one
end and has an anticodon
at the other end.
The anticodon acts to base
pair with the complementary
code on the mRNA
molecule, and delivers an
aa to the ribosome.
tRNA Structure and Function
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As tRNA reads the
mRNA transcript, it
brings an aa to the
ribosome and adds it
to growing
polypeptide.
The 2D shape is
similar to a cloverleaf.
2 Recognition Steps in Translation
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1. There must be a correct match between
tRNA and an aa.
2. The accurate translation of the mRNA
molecule.
1. The Correct Match
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1. Each aa gets joined to a
tRNA by aminoacyl-tRNA
synthase--there are 20 of
these, one for each amino acid.
This enzyme catalyzes the
attachment of aa to tRNA with
the help of some ATP energy.
The activated aa is now ready
to deliver the aa to the growing
polypeptide.
2. Accurate Translation
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The tRNA must correctly
match up the tRNA
anticodon with an mRNA
codon.
There is not a 1:1 ratio of
the tRNA molecules with
mRNA codons.
Some tRNA’s can bind to
more than one codon.
This versatility is called
“wobble.”
2. Accurate Translation
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Wobble enables tRNA to
bind differently in one of
its base pairs.
This is why codons for
some aa’s differ in their
3rd base.
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For example: the uracil at the 5’ end of a
tRNA anticodon can pair with an A or a G
in the third position of the 3’ end of the
mRNA codon.
Ribosomes
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These are the sites of
protein synthesis.
They consist of a large
and a small subunit and
are comprised of RNA
and protein.
 The RNA is ribosomal
RNA (rRNA).
 Bacterial (70s, 50S +
30s)
 Eukaryotic (80s, 60S +
40s)
Ribosomes
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rRNA genes are found on chromosomal
DNA and are transcribed and processed in
the nucleolus.
They are assembled and transferred to the
cytoplasm as individual subunits.
The large and small subunits form one
large subunit when they are attached to the
mRNA.
Ribosomes
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The structure of ribosomes fit
their function.
They have an mRNA binding
site, a P-site, an A-site and an
E-site.
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A-site (aminnoacyl-tRNA) holds
the tRNA carrying the next aa to
be added to the chain.
P-site (peptidyl-tRNA) holds the
tRNA carrying the growing
peptide chain.
E-site is the exit site where the
tRNAs leave the ribosome.
Each of these are binding sites
for the mRNA.
The 3 Stages of Protein Building
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1. Initiation
2. Elongation
3. Termination
All three stages require factors to help
them “go” and GTP to power them.
1. Initiation
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Initiation brings together
mRNA, tRNA and the 2
ribosomal subunits.
Initiation factors are
required for these things
to come together.
GTP is the energy
source that brings the
initiation complex
together.
2. Elongation
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The elongation stage
is where aa’s are
added one by one to
the growing
polypeptide chain.
Elongation factors are
involved in the
addition of the aa’s.
GTP energy is also
spent in this stage.
Recall from Chapter 5
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As the amino acids are being joined
together, the sequence and number of the
amino acids gives the protein its primary
structure.
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Recall from Chapter 5
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The secondary structure is forming
simultaneously as the hydrogen bonding
between the amino acids give a-helicies
and ß-pleated sheets.
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Recall from Chapter 5
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The tertiary structure is formed as more
amino acids are added and the R-group
interactions work to stabilize the protein.
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Recall from Chapter 5
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Lastly, the final functional protein structure
forms as multiple polypeptide chains join to
give the quaternary structure.
Not all proteins exist as multiple
polypeptides.
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3. Termination
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Termination occurs when a stop codon on the mRNA
reaches the “A-site” within the ribosome.
Release factor then binds to the stop codon in the “A-site”
causing the addition of water to the peptide instead of an
aa.
This signals the end of translation.
Polypeptide Synthesis
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As the polypeptide is being synthesized, it
usually folds and takes on its 3D structure.
Post-translational modifications are often
required to make the protein function.
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Adding fats, sugars, phosphate groups, etc.
Removal of certain proteins to make the protein
functional.
Separately synthesized polypeptides may need
to come together to form a functional protein.
Eukaryotic Ribosomes
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Recall the 2 types: Free and bound.
They function exactly the same and can switch
from free to bound.
This switch can occur when the protein that is
being translated contains a signal peptide
instructing the ribosome to attach to the ER.
Once attached to the ER, synthesis will continue
to completion and can then be exported from the
cell.
Signal Peptide Recognition
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The signal peptide is recognized as it
emerges from the ribosome by a proteinRNA complex called signal-recognition
particle.
The particle functions by bringing the
ribosome to a receptor protein built into the
ER where synthesis continues and the
growing peptide finds its way into the lumen.
Signal Peptide Recognition
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Once in the lumen of the ER, the newly synthesized
polypeptide is modified.
The signal peptide is cut out by an enzyme.
The protein then undergoes further processing and is
shipped where it needs to go.
Differences in Prokaryotic and
Eukaryotic Gene Expression
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Prokaryotic and eukaryotic RNA polymerases are
different, but perform the same function.
Transcription is terminated differently.
Prokaryotic and eukaryotic ribosomes are
different.
Transcription and translation are streamlined in
prokaryotes, it is compartmentalized in
eukaryotes.
Eukaryotic cells have a complex system of
targeting proteins for their final destination.