Download Chapter 17 Presentation

Chapter 17
From Gene to Protein
Main Questions:



The information content in DNA is the
specific sequence of nucleotides along the
DNA strands.
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?
Before Transcription and
Translation




Before these had been discovered, there was
many questions as to what caused many
metabolic errors.
In 1909, Archibald Garrod was the first man to
suggest that genes code for enzymes that dictate
phenotypes of organisms.
He called these “inborn errors of metabolism.”
He used alkaptonuria as a example.
George Beadle and Boris Ephrussi


Proposed a similar hypothesis regarding
eye pigment production in fruit flies.
As time went on, biochemists performed
numerous experiments that supported the
hypotheses of Garrod, Beadle and
Ephrussi.
Experiments Demonstrating the
Relationship Between Genes and
Enzymes



George Beadle and Edward Tatum experimented
with bread mold (N. crassa)
Bread mold is hearty. It can grow on minimal
medium because it can use various pathways to
synthesize everything it needs to survive.
Minimal medium is culture medium that contains
everything necessary for growth of the wild type:
usually inorganic salts, a carbon source including
vitamins and water.
Beadle and Tatum


Exposed the bread mold to X-rays to create a
variety of mutants.
Most mutants could survive on complete growth
medium--medium supplied with vitamins and
minerals.


Looked to see how they responded to various growth
medium--those lacking various nutrients required for
growth.
Looked closely at the pathway that synthesized
arginine.
Beadle and Tatum


Found that each mutant was unable to
carry out one step in the pathway for
synthesizing arginine because it lacked the
necessary enzyme.
They also noticed that the mutants would
survive if they were grown on a medium
supplied with a compound made after the
defective step.
Beadle and Tatum’s Famous
Experiment




They hypothesized the
arginine synthesis
pathway.
Examined 4 strains of N.
crassa.
Found mutants could only
grow when the compound
is supplied after the
defect.
Results confirmed their
one gene-one enzyme
hypothesis.
Beadle and Tatum


Somehow the gene that converts one
product to the next in the pathway must
have been affected by the X-rays.
One gene must be responsible for one
enzyme--their original hypothesis.
One-Gene One-Enzyme Hypothesis


As more was learned about proteins, it was
determined that not all proteins are
enzymes--(they are gene products, though)
The hypothesis was revised: It is now the
one gene-one polypeptide hypothesis.


Many enzymes are comprised of multiple
proteins.
Even this is wrong because some genes
code for RNA (that may not become
protein).
The Bridge Between DNA and
Protein



RNA is the single stranded compound that
carries the message from the DNA to the
ribosome for translation into protein.
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


Going from gene to protein.
Transcription is the synthesis of mRNA
using DNA as the template. Similar to DNA
synthesis.


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



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.
Polyribosome


Here you can see an
mRNA transcript being
translated into many
copies of protein by
multiple ribosomes in
a eukaryote.
This is a way in which
the cell can efficiently
make numerous
copies of protein.
Polyribosome


Here it is again in a
prokaryote.
The process
essentially the same
between prokaryotes
and eukaryotes.
One Main Difference


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



Scientists began wondering how the
genetic information contained within DNA
instructed the formation of proteins.
How could 4 different base pairs code for
20 different amino acids?
1:1 obviously didn’t work; a 2 letter code
didn’t work either; but a 3 letter code would
give you more than enough needed.
The Genetic Code



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




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



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




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



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


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


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


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




Promoters are found on the DNA molecule
and initiates the transcription of the gene.
This is the site where transcription factors
and RNA polymerase attach.
Transcription is finished when the RNA
polymerase reaches the terminator.
The stretch of DNA that is transcribed is
known as the transcription unit.
Promoters



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



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


In prokaryotes, RNA polymerase
recognizes and binds to the promoter on
the DNA 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


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



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

An electron micrograph showing the transcription
of 2 genes.
Transcription Termination


In prokaryotes transcription proceeds
through a DNA sequence that functions as
a termination signal causing the
polymerase to detach from the DNA.
This release of the transcript makes it
immediately available for use as mRNA.
Transcription Termination



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



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



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



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.


Introns--intervening regions
are removed.
Exons--expressed regions
are kept.
mRNA Modification



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?

In many genes,
different exons encode
separate domains of
the protein product.
RNA Splicing



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



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


tRNA , like mRNA, is
made in the nucleus and
is used over and over
again.
tRNA has an aa at one
end and an anticodon at
the other end. The
anticodon acts to base
pair with the
complementary code on
the mRNA molecule.
tRNA Structure and Function


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.
tRNA

tRNA is about 80
nucleotides long and
full of complementary
stretches of bases that
H-bond to one another
giving it a 3D shape
like an “L.”
tRNA

From this, the
anticodon loop comes
out of one end and at
the other end
protrudes the 3’ end
that carries the aa.
2 Recognition Steps in Translation


1. There must be a correct match between
tRNA and an aa.
2. The accurate translation of the mRNA
molecule.
1. The Correct Match



1. Each aa gets joined to
a tRNA by aminoacyltRNA synthase--there are
20 of these, one for each
amino acid.
This enzyme catalyzes the
attachment of aa to tRNA.
The activated aa is now
ready to deliver the aa to
the growing polypeptide.
2. Accurate Translation




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



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.
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





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



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


The structure of ribosomes fit
their function.
They have an mRNA binding
site, a P-site, an A-site and an
E-site.




P-site (peptidyl-tRNA) holds the
tRNA carrying the growing
peptide chain.
A-site (aminnoacyl-tRNA) holds
the tRNA carrying the next aa to
be added to the chain.
E-site is the exit site where the
tRNAs leave the ribosome.
Each of these are binding sites
for the mRNA.
QuickTime™ and a
TIFF (U ncompressed) decompressor
are needed to see this picture.
rRNA

rRNA is the most abundant form of RNA in
the cell because there are thousands of
ribosomes within a cell and about 2/3’s the
mass of a ribosome is rRNA.
The 3 Stages of Protein Building




1. Initiation
2. Elongation
3. Termination
All three stages require factors to help them
“go” and GTP to power them.
1. Initiation



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



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.
3. Termination



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


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.



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




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


The signal peptide is recognized as it
emerges from the ribosome by a protein-RNA
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



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.
3 Useful Properties of RNA



1. It can H-bond to other nucleic acids.
2. It can form a specific 3D shape by Hbonding on itself.
3. It has functional groups that allow it to
act as a catalyst.
Differences in Prokaryotic and
Eukaryotic Gene Expression





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.
Mutations



Mutations are changes in genetic material.
They can cover large portions of DNA or they can
be a point mutation.
There are 2 broad categories of point mutations:



Base-pair substitutions
Insertions or deletions
Mutations can be caused by a number of things:



Errors in replication, recombination, or repair
Mutagens--X, , UV radiation, chemicals
Spontaneous
Base-Pair Substitutions




One base pair gets substituted for another.
Sometimes these are silent because of the
redundancy of the genetic code.
Other times they are silent because the
mutation codes for an aa/protein that is not
essential to the protein’s function.
Also, there are times when the two aa’s
behave the same even though they are
different.
Missense Mutations Vs. Nonsense
Mutations


Missense mutations code for an aa, usually
the wrong aa.
In a nonsense mutation, the wrong aa is
encoded and it usually results in a stop
codon and the production of a nonfunctional protein.
Insertions and Deletions


These are often referred to as “frame-shift”
mutations because they alter the reading
frame of the mRNA.
They almost always result in a
nonfunctional protein.