Download Translation Tjian lec 26

Protein Translation
The genetic code
Protein synthesis Machinery
Ribosomes, tRNA’s
Universal translation: Protein Synthesis
Chemical Letters
DNA
A,T,G,C
mRNA
A,U,G,C
protein
*ribosome
*
A,C,D,E,F,G,H,
I,K,L,M,N,P,Q,
R,S,T,V,W,Y
20 synthetases
~45 tRNAs (E. coli)
1 tRNA <-> 1 amino acid
Amino Acid activation. The two-step process in which an amino acid (with its side chain denoted by R) is activated for protein
synthesis by an aminoacyl-tRNA synthetase enzyme is shown. As indicated, the energy of ATP hydrolysis is used to attach each
amino acid to its tRNA molecule in a high-energy linkage. The amino acid is first activated through the linkage of its carboxyl
group directly to an AMP moiety, forming and adenylated amino acid; the linkage of the AMP, normally an unfavorable
reaction, is driven by the hydrolysis of the ATP molecule that donates the AMP. Without leaving the synthetase enzyme, the
AMP-linked carboxyl group on the amino acid is then transferred to a hydroxyl group on the sugar at the 3’ end of the tRNA
molecule. This transfer joins the amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA
molecule. The synthetase enzyme is not shown in this diagram.
The genetic code is translated by means of two adaptors that act one after
another. The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino
acid to its corresponding tRNA molecule itself, whose anticodon forms base pairs with the
appropriate codon on the mRNA. An error in either step would cause the wrong amino acid to be
incorporated into a protein chain. In the sequence of events shown, the amino acid tryptophan (Trp)
is selected by the codon UGG on the mRNA.
Molecular models of valine and isoleucine. The extra methylene group in
isoleucine is marked. The synthetases specific for these amino acids are highly
discerning.
Glutaminyl-tRNA
synthetase complex.
The structure of this
complex reveals that the
synthetase interacts with
base pair G10:C25 in
addition to the acceptor
stem and anticodon loop.
A comparison of the structure of procaryotic and eucaryotic ribosomes. Ribosomal components are commonly designated
by the their “S values,” which refer to their rate of sedimentation in an ultracentrifuge. Despite the differences in the number
and size of their rRNA and protein components, both procaryotic and eucaryotic ribosomes have nearly the same structure and
they function similarly. Although the 18S and 28S rRNAs of the eucaryotic ribosome contain many extra nucleotides not
present in their bacterial counterparts, these nucleotides are present as multiple insertions that form extra domains and leave the
basic structure of each rRNA largely unchanged.
Head
30S
50S
CP
L11
A site
tRNA
Body
T.th. 70S
5.5 Å
h44
H69
Yusupov et al. (2001) Science 292, 883.
Formation of
the initiation
complex. The
complex forms in three
steps at the expense of
the hydrolysis of GTP
to GDP and Pi. IF-1, IF2, and IF-3 are
initiation factors. P
designates the peptidyl
site, A the aminoacyl
site, and E, the exit site.
Here the anticodon of
the tRNA is oriented 3’
to 5’, left to right.
Major Points
1. Alternative RNA splicing: one mechanism evolved to expand
diversity of gene products without increasing gene number
2. Control of alternative splicing by positive and negative splicing
factors ( analogous to transcriptional activators and repressors)
3. Complexity of organisms reflected by exon numbers and
differences between prokaryotic and eukaryotic mRNA’s
4. Universal triplet codon converts NA seq into amino acid seq:
total of 64 codes- AUG for Met and START, UAG,UAA&UGA
for STOP and the rest for the remaining 19 amino acids
5. Degeneracy of the code, ribosome entry site seq (Shine-Delgarno)
anti-codon seq in tRNA
6. High energy, amino-acyl-tRNA’s and specific synthetases
7. Importance of adaptor molecules (ie. tRNA synthetases and
anti-codon loop of tRNA’s)
8. Complex structure of ribosomes: protein and RNA components
Formation of the
initiation complex.
The complex forms in
three steps at the
expense of the
hydrolysis of GTP to
GDP and Pi. IF-1, IF-2,
and IF-3 are initiation
factors. P designates the
peptidyl site, A the
aminoacyl site, and E,
the exit site. Here the
anticodon of the tRNA
is oriented 3’ to 5’, left
to right.
Elongation cycle : binding of aminoacyl-tRNA, peptide-bond formation, and translocation.
First step in
elongation
(bacteria): binding of
the second
aminoacyl-tRNA. The
second aminoacyl-tRNA enters
the A site of the ribosome
bound to EF-Tu (shown here as
Tu), which also contains GTP.
Binding of the second
aminoacyl-tRNA to the A site is
accompanied by hydrolysis of
the GTP to GDP and Pi and
releaseof the EF-Tu•GDP
complex from the ribosome.
The bound GDP is released
when the EF-Tu•GDP complex
binds to EF-Ts, and EF-Ts is
subsequently released when
another molecule of GTP binds
to EF-Tu. This recycles EF-Tu
and makes it available to repeat
the cycle.
Second step in
elongation
(bacteria): formation
of the first peptide
bond. The peptidyl
transferase catalyzing this
reaction is probably the 23S
rRNA ribozyme. The Nformylmthionyl group is
transferred to the amino group
of the second aminoacyl-tRNA
in the A site, forming a
dipeptidyl-tRNA. At this stage,
both tRNAs bound to the
ribosome shift position in the
50S subunit to take up a hybrid
binding state. The uncharged
tRNA shifts so that its 3’ and 5’
ends are in the E site. Similarly,
the 3’ and 5’ ends of the
peptidyl tRNA shift to the P
site. The anticodons remain in
the A and P sites.
Third step in
elongation
(bacteria):
translocation. The
ribosome moves one
codon toward the 3’ end
of mRNA, using energy
provided by hydrolysis
of GTP bound to EF-G
(translocase). The
dipeptidyl-tRNA is now
entirely in the P site,
leaving the A site open of
the incoming (third)
aminoacyl-tRNA. The
uncharged tRNA
dissociates from the E
site, and the elongation
cycle begins again.
Termination of
protein synthesis in
bacteria. Termination
occurs in response to a
termination codon in the A
site. First, a release factor
(RF1 or RF2 depending on
which termination codon is
present) binds to the A site.
This leads to hydrolysis of
the ester linkage between
the nascent polypeptide and
the tRNA in the P site and
release of the completed
polypetide. Finally, the
mRNA, deacylated tRNA,
and release factor leave the
ribosome, and the ribosome
dissociates into its 30S and
50S subunits.
A polyribosome. (A) Schematic
drawing showing how a series of
ribosomes can simultaneously translate
the same eucaryotic mRNA molecule.
(B) Electron micrograph of a
polyribosome from a eucaryotic cell.
Ribosomes and endoplasmic reticulum. The electron micrograph and schematic
drawing of a portion of a pancreatic cell show ribosomes attached to the outer (cytosolic)
face of the endoplasmic reticulum (ER). The ribosomes are the numerous small dots
bordering the parallel layers of membranes.
Major Points
1. Multi-step protein synthesis : Initiation - small ribosome subunit
loads onto mRNA at the entry site and (AUG) START codon
along with IF1,2&3 followed by loading large subunit
2. Three distinct binding pockets/sites in the ribosome complex:
P- peptidyl ; A- aminoacyl ; and E - Exit sites
3. Several steps during protein synthesis require hydrolysis of GTP to
GDP & Pi (initiation complex formation; amino-acyl-tRNA binding
and translocation)
4. Peptidyl transferase reaction is catalyzed by ribozyme (RNA enzyme)
within the small ribosomal subunit (23S or 30S)
• Eukaryotic protein synthesis involves polyribosomes and a RNP
complex in which the 5’ Cap of mRNA is linked to the 3’ poly A
via various translation factors