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Section Q
Protein Synthesis
Q1 Aspects of Protein
Q2 Mechanism of Protein Synthesis
Q3 Initiation in Eukaryotes
Q4 Translation Control and
Post-translational Events
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Q1 Aspects of Protein
•
•
•
•
•
Codon-anticodon interaction
Wobble
Ribosome binding site
Polysomes
Initiator tRNA
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Codon-anticodon
Interaction
In the cleft of the ribosome, an anti-parallel
formation of three base pairs occurs
between the codon on the mRNA and
the anticodon on the tRNA.
The anticondon at one end of the tRNA
interacts with a complementary triplet
codon of bases on the mRNA, when
both are brought together in the cleft of
the ribosome.
The interaction is anti-parallel in nature.
Some highly purified tRNA molecules
were found to interact with more than
one codon, and this ability correlated
with the presence of modified
nucleosides in the 5’-anticodon position.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Wobble-I
• The wobble hypothesis
was suggested by
Francis Crick to explain
the redundancy of
genetic code.
• His specific predictions
are shown in right table
along with actual
observation.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Wobble-II
To explain the redundancy of
the genetic code. 18 aa are
encoded by more than one
triplet codons which usually
differ at 5’-anticodin base.
The 5'-anticodon base is able
to undergo more movement
than the other two bases and
can thus form non-standard
base pairs as long as the
distances between the ribose
units are close to normal.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Wobble-III
Base pairings at the wobble position
I: inosine
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Ribosome binding site
- Shine-Dalgarno sequence
• Only for prokaryotic
translation
• A purine-rich sequence
usually containing all or
part of the sequence 5'AGGAGGU-3'
• Locates 8-13 nt upstream
of the initiation codon in
prokaryotic mRNA
• Function: To position the
ribosome binding for
initiation of protein
synthesis
Section O: RNA Processing and RNPs.
Ribosome
Ribosome
mRNA
Yang Xu, College of Life Sciences
Ribosome binding site
- Shine-Dalgarno sequence
• John Shine is known to most
undergraduate biology students
for his role in defining the
Shine-Dalgarno gene sequence,
which is responsible for the
initiation and termination of
protein-synthesis.
Education
1972 BSc (Hons) 1st Class, Australian National University
1975 PhD, Australian National University
2006 DSc (Honoris causa) The University of New South
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Polysomes
Polysomes
• Each mRNA transcript is read by more than one ribosome.
• A second, third, fourth, etc. ribosome starts to read the mRNA
transcript before the first ribosome has completed the synthesis
of one polypeptide chain.
• Multiple ribosomes on a single mRNA transcript are called
polyribosomes or polysomes.
• Multiple ribosomes can not be positioned closer than 80 nt.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Polysomes
• Electron micrographs of ribosomes actively
engaged in protein synthesis revealed by
"beads on a string" appearance.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Initiator tRNA
fMet-tRNAfMet
The initiator tRNA fMet-tRNAfMet
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Q2 Mechanism of Protein Synthesis
• Overview
• Initiation
• Elongation
• Termination
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Overview
Three stages:
1. Initiation: the assembly of a ribosome on an mRNA molecule;
2. Elongation: repeated cycles of amino acid addition;
3. Termination: the release of the new protein chain.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Initiation
Steps
1. IF1 and IF3 bind to a free 30S subunit.
2. IF2 complexed with GTP then binds
to the small subunit. It will assist
the charged initiation tRNA to
bind.
3. The 30S subunit attaches to an
mRNA molecule marking use of
the ribosome-binding site (RBS)
on the mRNA.
4. The initiator tRNA can then bind to
the complex by base pairing of
its anti-codon with the mRNA.
5. The 50S subunit can now bind,
which displaces IF1 and IF2, and
the GTP is hydrolyzed in this
energy-consuming step.
6. 70S initiation complex formed at the
end of this phase.
Section O: RNA Processing and RNPs.
Initiation factors
30S subunit
1 2 3
G
G
2
fMet
Shine-Dalgarno sequence
3’
3 1
AUG
5’
Initiator tRNA
fMet
mRNA
3
G
30S initiation
Complex
5’
2
3’
1
G
1 2
50S subunit
P-site
A-site
3’
5’
70S initiation
Complex
Initiation in E coli
Yang Xu, College of Life Sciences
Elongation
Steps
1. Aminoacyl-tRNA delivery. EF-Tu is
required to delivery the aa-tRNA
to A-site. Energy is from GTP.
2. Peptide bond formation. After aatRNA delivery, the A- and P-site
are both occupied and the two
amino acids that are joined
closely. The peptidyl transferase
activity of the 50S subunit can
now form a peptide bond
between these two amino acids.
3. Translocation. A complex of EF-G
(translocase) and GTP binds to
the ribosome and the discharged
tRNA is elected from the P-site,
the peptidyl-tRNA is moved
from the A-site to P-site.
aa
aa
P-site
Empty A-site
3’
5’
aa
aa
aa
aa
Binding of aa-tRNA
to A-site
3’
5’
aa
aa
aa
Peptide bond formation
3’
5’
From A- to P-site
aa
aa
aa
3’
Section O: RNA Processing and RNPs.
5’
Yang Xu, College of Life Sciences
Elongation in E coli
EF-G:GDP
Aa-tRNA
Aa-tRNA-EF-Tu:GTP
EF-Ts
EF-Tu:GTP
GTP
EF-Tu:EF-Ts
EF-G:GTP
EF-Tu:GDP
GDP
EF-Ts
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Role of elongation factors in prokaryotes
• EF-Tu
–
–
–
–
It is a G protein (GTP/GDP-binding protein)
Active when GTP is bound (Kd = 10-6 M)
Inactive with GDP is bound (Kd = 10-8 M), slow offrate
Its off rate is increased by EF-Ts
• EF-Ts
– It is a guanosine nucleotide exchange factor (GEF) for
EF-Tu
• EF-G
– It is a G protein
– There is no known GEF for EF-G
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
N aa aa aa aa
aa
aa aa aa
aa
Termination
•
•
•
•
•
There are no tRNA species that
normally recognize stop codon.
Instead, protein factors called
release factors interact with these
codons. RF1 recognizes UAA
and UAG, and RF2 recognizes
UAA and UGA.
RF3 helps either RF1 or RF2 to
carry out the reaction.
The release factors mark peptidyl
transferase transfer polypeptide
to water rather than to the usual
aminoacyl-tRNA, and thus the
new protein is released.
To remove the uncharged tRNA
from P-site and release the
mRNA, EF-G is needed.
Section O: RNA Processing and RNPs.
Termination codon UGA
3’
5’
Release factor 1 or 2
G
N
aa aa aa aa
aa
aa aa aa
aa
Release factor 3
G
3’
5’
N
aa aa aa aa
aa
aa aa aa
Released polypeptide chain
Peptidyl transerase
aa
G
3’
5’
tRNA
Release factor
Yang Xu, College of Life Sciences
Termination
in E coli
Q3 Initiation in Eukaryotes
• Overview
• Scanning
• Initiation
• Elongation and Termination
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Overview
A comparison of the factors involved in prokaryotes and
eukaryotes is given in Table 1.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Differences between prokaryotes and eukaryotes
Bacteria
Eukaryotes
• Ribosome: 30S+50S →70S
• Few initiation factors (three factors)
• Ribosome: 40S+60S → 80S
• Many initiation factors (14 factors)
– IF1, IF2, IF3
• Elongation factors
– EF1A (EF-Tu), EF1B (EF-Ts), EF2
(EF-G)
• Release factors
– RF1, RF2, RF3
• Ribosome recycling factor
– RRF
• mRNA is not capped
• Direct binding of 30S particle next to
initiation codon (AUG) at ShineDalgarno sequence, 5’-AGGAGGU-3’
• Translation coupled to transcription
Section O: RNA Processing and RNPs.
– eIF1, eIF1A, eIF2, eIF2B, eIF3,
eIF4A, eIF4B, eIF4E, eIF4F,
eIF4G, eIF4H, eIF5, eIF5B, eIF6
• Elongation factors
– eEF1, eEF2
• Release factors
– eRF1 (or eRF2), eRF3
• Most mRNA is capped at 5’ end
and poly (A) at 3’ end
• 40S particle is recruited to 5’ cap
structure or poly(A) tail or an
internal ribosome entry site (IRES)
• Translation in always in cytoplasm
apart from transcription
Yang Xu, College of Life Sciences
Scanning
• Since there is no Shine-Dalgarno
sequence in eukaryotic mRNA, the
mechanism of selecting the start
codon must be different.
• Prof. Kozak proposed a scanning
hypothesis in which the 40S
subunit, already containing
initiator tRNA, attaches to the 5’end of the mRNA and scans along
the mRNA until it finds an
appropriate AUG.
• Eukaryotic small ribosome submit
complex bind to the 5’cap region
of the mRNA and moves along it
scanning for an AUG start codon.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Initiation
1.
Dissociation of ribosome
binding eIF1A (4C) and eIF3 to 40S
2.
Recruit ternary complex
(fMet-tRNAi, eIF2, GTP) to 40S
3.
Recruit mRNA to 43S → 46S particle
4.
46 S scans mRNA to reach AUG
5.
Initiation factors leave and 80S joins
6.
Elongation begins
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Initiation
1.
Dissociation of ribosome binding of
eIF1A (eIF4C-A-site) and eIF3 to 40S
•
•
•
2.
Recruit ternary complex (fMet-tRNAi,
eIF2, GTP) to 40S
•
3.
4.
5.
6.
eIF3 is a huge complex (MW=0.69
MDa) of 11 subunits not homologous
to IF3
Possibly eIF5, eIF5B and eIF1 bind at
this point as well
eIF1 seems to play a similar role as
IF3-C
•
•
eIF2 is a complex of three subunits (a,
, ) of 123 kDa
GTP is loaded by eIF2B
Regulated by phosphorylation
Recruit mRNA to 43S
•
mRNA is bound to the eIF4F complex
(4E, 4G, 4A)
Scan mRNA to reach AUG
•
Scanning requires a defined number of
eIFs
Initiation factors leave and 80S joins
•
•
eIF5 is the exchange GEF for eIF2→ eIF2
release
Ribosome is GEF for eIF5B→ eIF5B release
Elongation begins
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Elongation and Termination
Elongation
• The protein synthesis elongation cycle in prokaryotes and
eukaryotes is quite similar.
– Three factors (EF-Tu, EF-Ts and EF-G) are required properties
similar to their prokaryotic counterparts.
– eEF1α, eEF1βγand eEF2 have the roles described for EF-Tu, EF-Ts
and EF-G respectively.
Termination
• In eukaryotes, a single release factor, eRF, recognizes all
three stop codons and performs the roles carried out by RF1
(or RF2) plus RF3 in prokaryotes. eRF requires GTP for
activity, but it is not yet clear whether there is a eukaryotic
equivalent of ribosome release factor required for
dissociation of the subunits from the mRNA.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Q 4 Translation Control and
Post-translational Events
•
•
•
•
•
Translational control
Poly-proteins
Protein Targeting
Protein modification
Protein degradation
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Translational control
In prokaryotes
1. Short antisense molecules can obscure ribosome binding;
2. The formation of stems and loops can inhibit exonuleases;
3. The relative stability to nucleases of parts of the
polycistronic mRNA;
4. The binding of protein that prevent ribosome access.
In eukaryotes
1. Generally control protein amount by transcription of genes;
2. The repeats of the sequence 5’-AUUUA-3’ can make the
mRNA for rapid degradation and thus limited translation;
3. Protein binding can mask the mRNA (masked mRNA) and
prevent translation.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Polyproteins
• (1) Bacteriophage and viral
transcipts and (2) many mRNAs
for hormones in eukaryotes (e.g.
pro-opiomelanocortin) are
translated to give a single
polypeptide chain that is cleaved
subsequently by specific
proteases to produce multiple
mature protein from one
translation product. The parent
polypeptide is called polyprotein.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Protein Targeting-I
• Signal sequence
– It is a short peptide sequences which composed of about 13~36 amino acids.
– Function: The signal sequence causes the translating ribosome to bind
factors that make the ribosome dock with a membrane and transfer the
protein through the membrane as it is synthesized.
– Usually the signal sequence is then cleaved off by signal peptidase.
• Signal recognition particle (SRP)
– SRP can recognize ribosomes with signal peptide of the nascent chain.
• SRP receptor (docking protein)
– SRP with the arrested ribosome can bind to SRP receptor on the cytosolic
side of the Endoplasmic Reticulum (ER);
• Ribosome receptor protein
– When the ribosome becomes attached to ribosome receptor protein on the
ER, SRP is released and can be re-used.
– The ribosome is able to continue translation, and the nascent polypeptide
chain is pushed through into the lumen of the ER.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Protein Targeting-II
• Glycosylation
– The protein in ER is usually modified by glycosylation, and different
patterns of Glycosylation seem to control the finial location of the protein.
• Nuclear localization signal (nls)
– Different N-terminal sequences can cause protein to be imported into
mitochondria or chloroplasts, and the internal sequence –Lys-Lys-LysArg-Lys, or any five continuous amino acids, can be a nuclear localization
signal (nls). It cause the protein to be imported into the nucleus.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
The secretory pathway
in eukaryotes (co-translational targeting)
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Protein modification
The most common alterations to nascent polypeptides
are those of cleavage and chemical modification.
1. Polypeptide cleavage (by amino- & carboxypeptideases)
•
•
•
•
2.
To remove signal peptides;
To release mature fragments from polyprotein;
To remove internal peptides as well as
To trim both N- and C-termini.
Chemical modification:
•
•
•
•
•
Acetylation;
Hydroxylation;
Phosphorylation;
Methylation;
Glycosylation.
Phosphorylation
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Protein degradation
A protein that is damaged, modified or has an inherently destabilizing N-terminal
residue becomes ubiquitinylated by covalent linkage of molecules of the small,
highly conserved, ubiquitin, via its C-terminal Gly, to lysine residues in the
protein. The ubiquitinylated protein is digested by a 26S protease complex in
a reaction that requires ATP and releases intact ubiquitin for re-use.
In eukaryotes, N-terminal residue plays a critical role in inherent stability:
• t1/2>20 hours: Ala, Cys, Gly, Met, Pro, Ser, Thr, Val (eight aa);
• t1/2 2~30 min: Arg, His, Ile, Leu, Lys, Phe, Trp, Tyr (eight aa);
• Destabilizing: Asn, Asp, Gln, Glu. (4aa)
26S protease complex
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
That’s all for Section Q
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences