Download TRANSCRIPTION-TRANSLATION PROTEIN SYNTHESIS

TRANSCRIPTION-TRANSLATION
PROTEIN SYNTHESIS
BEGINS
Somethin’ weird
In the neighbourhood
Who you gonna’ call?
Protein Synthesis History
• Garrod (1902) one gene one enzyme
hypothesis
• Beadle and Tatum(1935-1944) one gene one
polypeptide hypothesis
• Vernon Ingram gene specifies the kind and
location of each amino acid in a polypeptide
chain.
• RNA links DNA to protein sequence
• Crick’s central dogma
Early 20th century geneticists were faced with several
problems:
1. What are genes?
2. How do genes work?
3. What produces the unique phenotype associated with a
specific allele
Yea, 20th century
scientists
We’re in the
21st century
you know.
Back off man, we’re
scientists
The ONE GENE ONE PROTEIN HYPOTHESIS.
Garrod a physician 1909
postulates
Hey, what kind of urine sample is this? It’s not
like the others with the amber colour. How
can the urine be black? I want to investigate
that.
Black Urine. That’s
when you call....
I tested the urine and I found it contained alkapton.
Alkapton turns black on contact with air and it was
causing the black urine colour. I furthered the study
and discovered the chemical was in the blood of the
patient as well. This led me to believe that it was an
inborn (genetic) error in metabolism. By the way, I
determined it was an autosomal recessive gene
My hypotheses were
1.Individuals with alkaptonuria have a defective gene,
making them unable to produce the correct alkaptonmetabolizing enzyme that most people possess.
2. The enzyme is produced as a result of hereditary
material. Defective hereditary material leads to
defective enzyme.
A
Enzyme 1
B
Enzyme 2
C
Enzyme 3
B
Initial
reactant
Intermediate metabolites
B
B
B
Final
product
EXAMPLE
If enzyme 2 is defective,
substance B will
accumulate
One gene, one enzyme hypothesis - A single
gene controls the specificity and activity of
each enzyme in a given metabolic pathway
MEANING- mutation of this gene changes the
ability to the cell to carry out a particular
reaction, thus disrupting the pathway.
D
Next on the scene is Beadle and Tatum They demonstrated
experimentally Garrod’s hypothesis to the one gene one
enzyme and changed it to the one gene one polypeptide
Hypothesis.
when TATUM
Shhh. Don’t say
BEADLE S
fungus, most
battle with paddles people associate
in a puddle,
it with my
athlete’s foot
they call it a tweetle
beetle puddle
paddle battle.
AND...
When beetles...
We used
fungus in our
experiment!
The Beadle and
Tatum
EXP is great!
STEP 1
CAUSE the MUTATION
STEP 2
Take samples that will not grow on minimal
medium like the normal Neurospora, and
grow them on enriched These are the
mutants- but for what protein?
STEP 3
Switch cultures from enriched medium to
minimal medium with a particular
supplement. If you have growth of the
culture, this identifies the mutant strains
that require the stated nutrient
One gene one polypeptide Revision of the earlier one
gene one enzyme hypothesis . Some proteins are made up
of multiple polypeptide chains and these are coded by
separate genes.
MEANING- a mutation in a gene coding for a specific
polypeptide can alter the ability of the resultant protein to
function. This can result in an altered phenotype of the
individual!
Guys these are not enzymes affected by mutations.
These are one of the polypeptides that makes up the
protein hemoglobin. Lets go see my friend Vernon!
Just look
at those
filthy
mutated
blood cells
I’d recognize
those sickle
shaped blood
cells
anywhere!
Isn’t this guy too old to work
with us?
Relax, the guy is a genius. He is
considered the FATHER OF
MOLECULAR MEDICINE. He figured
out that sickle cell anemia is
caused by a gene with a defect for
a single amino acid
He’s right , you know.
All of our advances
stand on the
foundations of these
dedicated scientists
work; especially the
old ones.
Dr. Vernon Ingram linked a human hereditary
abnormality(sickle cell anemia) to a single alteration of an
amino sequence in the protein. This is a single defect in
the genetic code that is expressed by the individual.
Mutated Sickle gene
Normal gene
Remember
me? I
helped
support
this effort
Dr. Ingram and others determined that the hemoglobin
molecule of sickle cell disorder was due to the substitution
of glutamic acid in position 6 of the β chain. Ingram used
electrophoresis and chromatography to show the
difference between the normal and mutated gene .
This is the machine for gas
chromatography. (not paper
chromatography like we did) It
separates out chemicals based on
molecular character. It’s a highly sought
skill if you can work one of these. CSI
kinda stuff!
Electrophoresis
Check us out
Get trained here in gas chromatography.
We actually have the machines you can
train on, unlike some local universities!
A
enzyme1
B
enzyme2
C
enzyme3
D
The above diagram corresponds to a polypeptide pathway. A, B,C,D
are all represented in the graph below and your job is to determine
which colour each of the letter boxes is from the experimental
evidence of different mutants of bacteria growing on different media.
MUTANT
GROW ON
RED
GROW ON
BLUE
GROW ON
PURPLE
GROW ON
BLACK
1
GROWTH
NO GROWTH
NO GROWTH
GROWTH
2
GROWTH
NO GROWTH
GROWTH
GROWTH
3
NO GROWTH
NO GROWTH
NO GROWTH
GROWTH
Mutants will
lack enzymes
1 2 3?
Red must
be third as
2 varieties
grow on it
Blue must be
first as none
of the
mutants can
grow on it. You
need all three enzymes
to get to D
Purple
must be
second as
only1
variety
grows on it
Black must be
last as all
varieties
survive on it.
You don’t need
enzymes if it is
given
RNA is the link between the DNA and the
protein sequence
Scientists for some time had suspected such
a link between DNA and proteins
Evidence pointed to the link being the RNA
molecule as RNA is found is not restricted
to the nucleus like the DNA.
RNA is also found on the ribosomes in the
Endoplasmic reticulum
Now it’s time for me to discuss my
Central Dogma
It’s not so bad.
Think of
Einstein. He
did his best
stuff when he
was a patent
clerk. You don’t
have to be like
Francis Crick
and work at
Cambridge
University.
It’s a major
disgrace losing
our positions at
the university.
Forget MIT or
Stanford now.
Personally, I liked working for
the university! They gave us
money and facilities. We
didn't have to produce
anything. You've never been
out of college. You don't
know what it's like out there!
I've worked in the private
sector... they expect results
The central dogma states information
flow is from DNA to RNA via
transcription and thence to protein via
translation.
DID YOU KNOW?
Only about 3% of
the human
genome is both
transcribed and
translated. This
means a vast
amount of the
DNA is not
apparently used!!
THE SECRET CODE OF DNA
Transcription makes a
Translation makes polypeptides
copy of DNA on the mRNA
from the mRNA codes.
In prokaryotes, shown
here, the promoter is
needed to bind RNA
polymerase
The side of the DNA
the promoter (P) is
on is the same side
of the DNA that will
act as the template
strand.
I am the
end!
T
P
The
terminator(T)
signals the
end of
transcription
You can see from the diagram that the promoter is upstream and the terminator
downstream. The yellow arrow indicates the copy direction.
DNA has code which is like a language. It stores vast amounts of
information utilizing only 4 different chemicals ATCG for short.
1.DNA is transcribed to mRNA which is the chemical that is to be read like a
language. It has the letters AUCG instead of our 26 letter alphabet
2. You have to know what direction to read in. English is right to left but
Arabic is left to right. RNA is read in a 5’ to 3’ direction
3.Words in languages are separated by spaces. There are no spaces in RNA so
each “word” is called a codon and is three nucleotides in length.
4.Sentences begin with a capital to indicate a start. In codon language starts are
indicated by AUG which is the code that translates to the amino acid methionine
5. Sentences indicate a completion of information with a period. RNA indicates
the end of the code with a stop signal, which are specific codons.
The secret code of DNA involves the use of 4 different letters in
codons that are 3 nucleotides long. These must code for the 20
amino acids.
Why aren’t the codons only two long or four long.
Why are they always 3 nucleotides long?
Mathematically in order to determine how many
different codes can be made using only two letter
code you would get 42= 16 which is not enough to
code for the 20 amino acids.
If you go to the next smallest letter (3 letters) code
you get 43 = 64. This will accommodate all 20 amino
acids, allow for some stop signals and allow for some
repetition of letters that will form the same amino
acid . Ie – Allows for some errors without errors in
the amino acid determined in TRANSLATION.
This is the
strand that
you use to
make the
mRNA
copy. It is
called the
template
strand or
antisense
strand.
You can see from this layout that you are ready to translate the
RNA code. Remember the codons are always three nucleotides
long. The secret code must be decoded utilizing the mRNA codon
chart on the next slide.
met
pro
leu
asp
arg
stop
Once you hit the stop
codon you STOP and
the polypeptide is
finished even though
there may mo more
codons
Follow the
coloured
boxes to
translate
the
codons
We noticed that for many of the codons, they
code for the same amino acid. Most allow for a
mistake on the third nucleotide of the codon.
WOBBLE
ANTICODONS CAN PAIR WITH
MORE THAN ONE CODON AS A
RESULT!!
The Wobble hypothesis
states that there are
synonymous codons for
a given amino acid
usually differing in their
third base. WOBBLE
enables the third
nucleotide of some
tRNA anticodons to pair
with more than one kind
of base in the codon.
EUKARYOTIC PROTEIN SYNTHESIS
promoter
TATA
In eukaryotes, the
promoter, several
transcription
factors and usually
a TATA box are
needed to initiate
transcription
complex
Problem here. You
have a brain and
can use the chart
to decipher the
codons; the cells
don’t. So how do
they do it?
The first step, called TRANSCRIPTION, is initiated in the nucleus of eukaryotic cells.
Primary transcript OR
transcription unit produced
by RNA polymerase II
3’
5’
As RNA polymerase II copies it is
called ELONGATION
Introns
removal
RNA
polym
erase
Post transcriptional
modification removes
introns leaving exons
which are joined
together.
Spliceosomes remove introns with snRNA snRNP
Exons spliced together
5’ cap
Coding part
Poly A tail
During transcription
the 5” end is capped with modified G
the 3’ end is modified with a poly A tail.
Note: RNA polymerase II
is used in EUKARYOTIC
transcription. RNA
polymerase I and II are
not translated
The mRNA transcript now leaves the nucleus through the nuclear pores and enters
the cytoplasm for the TRANSLATION STEP
EUKARYOTIC TRANSCRIPTION
TERMS: Upstream begins downstream at end
Remember that in the transcription process, the RNA polymerase only copies the
template(antisense) strand to mRNA in a 5’ to 3’ direction. You may notice,
however, that the mRNA strand is almost the same as the DNA copy (sense) strand
only with all the T replaced by U.
SPECIAL NOTE: TRANSCRIPTION
Eukaryotes have three different RNA
polymerases for transcription
RNA polymerase I makes ribosomal RNA
RNA polymerase II makes mRNA genes that code
for protein
RNA polymerase III makes transfer RNA
Transcription occurs one nucleotide
at a time
RNA polymerase does NOT
proofread
TRANSLATION – THE PLAYERS
ala
met
gly
thr
val
In the cytoplasm where
translation takes place,
All these RNA
molecules are
synthesized in the
there are 3 different types
of RNA. Messenger
mRNA with the codons,
ribosomal rRNA which
together with a little
protein makes the
ribosome, and transfer
tRNA with the anticodons
and attached amino acids
nucleus by RNA
polymerase.
Nucleoli show
high transcription
40s
60s
This ribosome is a eukaryotic variety
called 80s. It has a 40s and 60 s
subunit (sorry they don’t add up)
DID YOU KNOW?
We have our own DNA and
ribosomes, but both are more similar
to prokaryotic cells!
The mitochondria and chloroplasts have their own
ribosomes and these are 70s, similar to those found
in bacteria of today. This supports the
endosymbiotic theory of the origin of these
organelles.
Antibiotics like tetracycline and streptomycin
target the bacterial ribosomes 70s but do not
harm the 80s variety of eukaryotes. The 70s
ribosomes of mitochondria are not harmed
because they are inside the cell protected by a
double membrane.
Recent evidence concludes that the functional
part of the ribosomes are made of RNA. This
again supports the concept of the ribozyme- RNA
acting as both an enzyme to catalyze a reaction
and also storage of hereditary information.
Listen, you just have to
check out my presentation
on origin of First Cells in
the evolution section.
Learn about RNA evolution
there!
The anticodon will match the codon
sequence and thus direct this tRNA
carrying the amino acid glutamic acid to
the correct region to be attached
Glutamic acid is
electrically
charged. It’s
obvious because
it’s an ACID!
Acids ionize
glu
Each transfer RNA becomes charged with the
appropriate amino acid by the enzyme aminoacyl
synthetase. In the process an ATP is used up and
converted NOT to ADP but rather AMP!!!
The ribosome has three attachment
points for the tRNA and forms the
peptide bonds.
The A site is the
ATTACHMENT site for the
tRNA.
E
site
P
site
A
site
The P site is the PEPTIDE site
and it is where the peptide
bonds are formed between
the amino acids
The E site is the EXIT site, where the tRNA is
released to be recharged and recycled.
Note : Two of these these names are simplified for better understanding . A site is aminoacyl
binding site , P site is peptidyl binding site
TRANSLATION
met
pro
leu
asp
arg
TRANSLATION is accomplished
by a method similar to that
animated here. One omission
is that each tRNA must be
shown binding only when the
ribosome moves and makes the
A site available.
GTP is hydrolyzed in this process
ANIMATION SHOWS THE PROCESS OF
TRANSLOCATION
PROKARYOTE PROTEIN SYNTHESIS
In prokaryotes, the process
is similar but there is no
post transcriptional
modification. The bacterial
genes have no introns.
Since prokaryotes have no
nucleus, transcription and
translation can occur at the
same time as shown in the
diagrams.
Ribosomes are
smaller in
prokaryotes.
They are 70s.
PROKARYOTE PROTEIN SYNTHESIS cont’d
When the stop signal is
encountered, the
ribosome disengages
from the mRNA.
The ribosomes of
prokaryotes have a 30s
subunit and a 50s
subunit. Overall they are
called 70s (sorry they
don’t add up)
50s
30s
POLYRIBOSOMES –
several ribosomes
copy the mRNA at the
same time.
Now the primary structure
for the polypeptide is
formed, the next phase
involves chaperone
proteins that assist the
proteins in the folding
process to form the proper
shape. They can prevent
proteins from denaturing
in the folding process.
A chaperone protein ?
What is this? Some
1950’s prom?
Well that pretty much
explains the whole
process of protein
synthesis. We’ll bill
ya.
TRANSCRIPTION-TRANSLATION
PROTEIN SYNTHESIS
ENDS