Download BCH401G Lecture 39 Andres Lecture Summary: Ribosome

BCH401G
Lecture 39
Andres
Lecture Summary:
Ribosome: Understand its role in translation and differences between
translation in prokaryotes and eukaryotes.
Translation: Understand the chemistry of this process, the proteins that
are used and their individual roles.
Which steps are regulatory? Look at the steps that require energy (GTP
or ATP hydrolysis)-why/how is this energy used. Understand how to
calculate energy use.
What is the relationship between translation rate and fidelity?
How would mutations in individual proteins alter the process of
translation (think back to our discussion of tRNA charging)?
Overview of Translation:
What are key players in translation? We have already talked about
two of these:
1. mRNA
2. tRNAs charged with each of the amino acids
We need one more thing:
3. The Ribosome. The Ribosome is where the process of
translation occurs.
Ribosomes are very large complexes composed of rRNAs and proteins.
In E. coli the large subunit is termed the 50S subunit. The Small subunit
is the 30S subunit.
50S subunit and 30S subunit associate to form a 70S ribosome.
1. 50S subunit. Complex of two rRNAs, 23S rRNA and 5S rRNA, and 31
different proteins.
2. 30S subunit. Complex of 16S rRNA and 21 different proteins.
How does the ribosome perform the translation of mRNA to
protein?
1. Provides a place where codons of mRNA can interact with
tRNAs carrying the amino acids designated by the codons.
Looks simultaneously at two codons of mRNA:
5' -------------------GGCGCA------------- 3'
Gly Ala
2. Then it catalyzes formation of a covalent bond between the
amino acids of tRNAs which are base-pairing to adjacent
codons on the mRNA.
The bond between amino acids is called the "Peptide Bond".
A Peptide Bond is formed between the carboxyl group of one
amino acid and the Alpha amino group of another amino
acid.
mRNAs have a 5' end and a 3' end - they have Polarity.
Proteins also have polarity.
1. The Amino acid at one end of a protein chain has a free alpha amino
group.
Called "Amino-Terminus" or "N-terminus" of the protein.
2. Amino acid at other end has a free Alpha carboxyl group.
Called "Carboxy-Terminus" or "C-terminus" of the protein.
Direction of Protein Synthesis is from N-terminus to C-terminus.
Each new amino acid is added onto the C-terminal end of growing
protein chain.
Alpha amino group of the new amino acid (bound to a tRNA)
attacks the carboxyl group of the last amino acid in protein
chain.
By convention, amino acid sequences are written and numbered
left-to-right from N-terminus to C-terminus.
Initiation of Translation
Have a mRNA molecule:
5' ---------------------------------------------------------- 3'
Ribosome wants to translate the mRNA into protein. But there are two
problems:
Problem 1: What reading frame should be used?
In any mRNA sequence, there are multiple ways (three reading frames)
codons could be read.
Each way to read the codons is called a "Reading Frame". Three
possible "Reading Frames"
Very important for the ribosome to find the correct reading frame.
If wrong frame is used -Generate a protein with the wrong amino acid
sequence. Not functional.
2. Problem 2: At what codon in the mRNA does the ribosome
begin translation?
Turns out both of these problems are solved in the same way.
The solution is that the ribosome begins translation at a specific AUG
codon within the mRNA template termed the "Start Codon".
This is a methionine codon, so the first
amino acid in proteins is usually
methionine.
In E. coli the first methionine is modified
with a formyl group.
Called formyl-methionine (fMet). This
special fMet is attached to a unique
initiator tRNA (tRNAfMet).
All subsequent methionines in the protein
do not have a formyl group. Only the
initiator methionine is modified.
But how does the ribosome recognize this particular AUG codon?
There are many potential AUG codons in each mRNA sequence.
Sequences within the 16S rRNA, which is part of the small subunit of the
ribosome, interact with a complementary sequence in the
prokaryotic mRNA near the AUG Start Codon.
This sequence in the mRNA is called the "Shine-Dalgarno
Sequence"
Interaction of this sequence with the 16S rRNA causes the ribosome to
begin translation at this particular AUG codon.
You can see that starting translation at a specific AUG codon also
solves the Reading Frame Problem.
Next three nucleotides encode the second codon, and so on.
1. The Shine-Dalgarno sequence in the mRNA tells the ribosome
to initiate translation at a nearby AUG codon.
2. This AUG codon is the first codon of the proper reading frame.
Reading frame is now set.
This also allows prokaryotes to generate mRNAs that encode multiple
proteins (operons). Each protein coding region would begin with a S.D.
sequence which allows the individual coding regions to be translated.
This is not the case for Eukaryotes.
Steps of Initiation of Translation.
1. Small subunit (30S) interacts with proteins called Initiation
Factors.
Initiation Factors help assemble the Initiation Complex.
IF-1, IF-2, and IF-3. IF-2 contains a bound GTP nucleotide.
Initiator tRNA (tRNAfmet) carrying
a formyl-methionine joins the
initiation complex.
Then mRNA joins the complex. The
16S rRNA of the small subunit
identifies the correct AUG Start
Codon.
The Anticodon of the Initiator tRNA
(fMet) then base-pairs with the
AUG Start Codon.
This is called the "30S Pre-Initiation
Complex".
2. The Large subunit (50S) then associates with the Pre-Initiation
Complex.
Initiation Factors are then released.
IF-2 release requires the hydrolysis of the Gamma Phosphate of
the bound GTP, converting it to GDP. The phosphate bonds of
many nucleotide triphosphates are used to drive the process of
translation. Hydrolysis of these bonds liberates energy that can be
used to do work.
In this case, GTP hydrolysis
causes release of IF-2 (drives
a change in conformation)
from the Initiation Complex.
The joining of the 50S subunit with
the"30S Pre-Initiation Complex"
gives the "70S Initiation
Complex". The Ribosome is now
ready to begin synthesizing an
amino acid sequence from the
mRNA template.
Elongation of Translation.
To understand how each amino
acid is added to the protein chain,
we must look more closely to the
region of the ribosome where this
process is occurring.
Ribosome only looks at two codons
of mRNA at any one time.
Ribosome can only interact
with two tRNAs at any single
time. One tRNA for each
codon.
The sites where these two tRNAs bind are called the "peptidyl" site
or "P" site and the "aminoacyl" site or "A" site.
The Peptidyl site contains the tRNA that is attached to the growing
chain of amino acids.
This tRNA is called the Peptidyl tRNA. A short chain of amino acids is
called a "Peptide". This is why this site is called the "Peptidyl tRNA" and
"Peptidyl" site.
The "A" site is where the charged tRNA carrying the next amino acid
binds to ribosome.
Three steps in Elongation of Translation:
1. Binding of the appropriate charged tRNA to the "A" site.
2. Peptide Bond Formation between the amino acid on the tRNA in
the "A" site and the growing amino acid chain attached to the
tRNA bound in the "P" site.
3. Translocation of the Ribosome.
The "A" site tRNA (now carrying amino acid chain) moves to the "P"
site. A new"A" site can now be created.
The Ribosome moves one codon down the mRNA.
These processes involve "Elongation Factors" or "EFs". These
proteins assist in the elongation steps of translation.
Step 1. Binding of charged tRNA to the "A" site.
This involves an elongation factor called EF-Tu.
EF-Tu-GTP forms a complex with charged tRNAs.
Like IF-2, EF-Tu also has bound GTP nucleotide. Will write it as
EF-Tu-GTP
First, the charged tRNA bound to EF-Tu-GTP enters the "A" site.
Another name for a charged tRNA is an aminoacyl tRNA.
This is why it's binding site on the ribosome is called the aminoacyl site.
The ribosome must then examine the different charged
tRNAs that diffuse into the "A" site until it finds one with an
anticodon that base-pairs correctly with the codon found in
the mRNA template.
When binding of a proper tRNA is verified by correct WatsonCrick base-pairing to the codon, EF-Tu releases the
charged tRNA. The timing of this period of analysis is
dictated by the rate of GTP hydrolysis and is therefore
regulated by EF-Tu (GTPase).
The energy for the dissociation of EF-Tu from the charged tRNA comes
from the hydrolysis of the bound GTP to GDP + Pi.
So now we write this as EF-Tu-GDP, which is now released from
the ribosome complex.
A peptide bond can not be formed until EF-Tu is released from the
charged tRNA. It takes time for hydrolysis to occur and for EF-Tu
GDP to leave the ribosome. During either period an incorrect
charged-tRNA can be released from the A site, whereas a correct
tRNA will remain bound.
There is a balance between the time that EF-Tu remains bound, and its
rate of release. A very long binding time would result in very accurate
protein synthesis but at a very slow rate, rapid release would result in
fast but inaccurate protein synthesis (lots of protein but none of the
enzymes would be active due to errors).
EF-Tu is then needed to initiate a new cycle of elongation used by
binding to another charged tRNA, but first the GTP-bound form must be
regenerated by the action of another Elongation Factor called EF-Ts.
EF-Ts causes GDP to be released from EF-Tu and replaced with
GTP. This action is termed "guanine nucleotide exchange" or "
guanine nucleotide dissociation stimulation" so EF-Ts is a
nucleotide exchange factor or dissociation stimulator for EF-Tu.
Step 2. Peptide Bond Formation.
Once the correct charged tRNA
is present at the "A" site, a
peptide bond is formed
between the amino acid carried
by the tRNA and the growing
amino acid chain attached to
the tRNA bound in the "P" site.
Peptide bond formation is
catalyzed by the peptidyl
transferase activity site of the
ribosome (nucleophilic attack
of the amino nitrogen on the
ester high energy bound
formed originally by the
aminoacyl-tRNA synthase of
the peptidyl tRNA).
Alpha amino group of the new
amino acid attacks the alpha
carboxyl group found on the
growing peptide chain.
As a result, the growing
amino acid chain is now
attached to the tRNA
found in the "A" site and
the chain has been
extended by one amino
acid.
Step 3. Translocation.
Translocation involves a third elongation factor called EF-G which
also contains a bound GTP. Written as: EF-G-GTP.
After Peptide Bond Formation, hydrolysis of the bound GTP to GDP on
EF-G causes two things to happen:
1. The "A" site tRNA (now carrying amino acid chain) moves to the
"P" site.
The "A" site is now open.
2. At the same time, the Ribosome moves one codon down the
mRNA molecule.
A new charged tRNA can now enter the open "A" site and interact
with the next codon in the mRNA. So the GTP hydrolysis on EF-G
allows the coordinated movement of the ribosome complex which
serves to open a new codon site and allows translation to
continue.
The Ribosome simply repeats these three steps as each new
amino acid is added to the peptide chain.
The rate of Protein Synthesis is approximately 18 a.a./second.
Termination of Translation
Termination involves Proteins called Release Factors: RF-1 RF-2.
and RF-3.
Elongation of translation
occurs until the ribosome
reaches a Stop Codon (UAA,
UAG, UGA).
At this point a Stop Codon is
present at the "A" site.
The problem is that the cell
does not contain a charged
tRNA that is able to interact
with these Stop codon
sequences.
Since there are no tRNAs that
contain the proper anticodon to
base-pair with the Stop Codons,
extension of the peptide chain is
halted.
Instead, RF-1 or RF-2 bind
to the Stop Codon.
RF-1 binds and recognizes
either UAA or UAG codon
sequences; RF-2 binds to
UAA and UGA stop
codons.
RF-3 interacts with RF-1 or
RF-2 and RF-3 also binds
GTP: RF-3-GTP
Binding of the Release Factors to the Stop Codon leads to the
hydrolysis of the amino acid chain from the Peptidyl tRNA in the "P" site.
GTP bound by RF-3 is hydrolyzed to GDP. This causes a
conformational change in the RF-3 protein and this causes the
Release Factors to dissociate from the Ribosome.
Synthesis is complete. The newly synthesized protein leaves the
ribosome.
Energy Cost of Translation. Translation requires a large amount of
energy from the cell.
For each amino acid added to the chain, ~4 high energy
phosphate bonds are broken.
2N
ATP is cleaved to AMP + 2 Pi during charging of the tRNA
with a specific amino acid. Two bonds.
1
GTP for initiation.
N-1
N-1
GTPs are required for the formation of N-1 peptide bonds.
GTPs are necessary for the N-1 translocation steps.
1
GTP is required for termination.
This does not include any proofreading and is therefore a
conservative estimate.
For a typical 300 amino acid protein: 50,000 kJ energy/mole.
Translation is a very energy intensive process (single peptide
bond +20 kJ/mole) because the order of amino acids is critical as
is a high degree of fidelity [ 20300 different possible ways to put 20
amino acids together to give a 300 aa long peptide].
In some cells, it has been estimated that up to 80% of total
available energy (ATP/GTP) is used in protein synthesis.
This is why gene expression is so carefully regulated
(transcription, translation).
Cell loses a lot of energy if it expresses genes it doesn't need.
Antibiotics. Antibiotics are used to fight bacterial infections.
Many antibiotics inhibit bacterial growth by inhibiting the process of
translation.
Inhibition specific to
translation in bacteria, not
eukaryotic cells. So they kill
the bacteria infecting you
without killing you.
Examples:
Tetracycline binds to 30S
subunit and prevents
binding of charged tRNAs to
"A" site.
Erythromycin binds to 23S
RNA and inhibits the
Translocation Step,
blocking elongation.
The problem with using
antibiotics is that microorganisms
can develop resistance. This
often results from the acquisition
of a particular "resistance gene".
Erythromycin resistance is
gained by the expression of a
methylase enzyme that
inactivates the drug by blocking
its ability to bind to the 23S RNA.
Antibiotic resistance is used as a
tool in molecular biology (we will
see how in the next lecture).
Differences between
Prokaryotic and Eukaryotic Translation:
1.Eukaryotic Ribosomes are larger and require 4 rRNAs.
2.The initiator tRNA is methionine (not fMet), however as with
prokaryotes, the tRNA is unique and used only to initiate
translation.
3.Start signal. No purine rich recognition site, usually the first AUG
codon is used. The 5' cap is used to initiate translation and
because only one 5' AUG is selected, any single mRNA can
produce only a single protein.
4. Many more initiation factors are used.