Transcript Document

Chapter
17
Protein Synthesis , Folding and
Processing
mRNA directed protein biosynthesis is also
called translation in molecular biology.
Why ?
Protein biosynthesis is a process to translate
information from the nucleotide sequence of an mRNA into the
sequence of amino acids of the corresponding specific protein.
Section One
Components Required for Protein Synthesis
The process of protein synthesis is one of
the most complex events in the cell.
It involves the coordinated participation of
over 100 biomolecules.
Biomolecules Required in Protein Synthesis
mRNA
the template
tRNAs
transferring amino acids
ribosome the location of protein synthesis
aminoacyl-tRNA synthetases link tRNA and
amino acid
protein factors
ATP and GTP
inorganic ions
A
mRNA works as the template in protein
synthesis.
the structure of a eukaryote mRNA
Cap-----AUG-------------stop codon--------poly A
5’UTR coding region
3’UTR
What is the relation between the coding
region in mRNA and the amino acid se-quence in
the corresponding protein?
The Genetic Code
The genetic code is the way in which the nucleotide sequence in mRNA ( or DNA ) specifies the amino acid sequence in protein.
How ?
A, G, U and C are organized into triple-nucleotides called codons.
There are 64 ( 4x4x4 ) codons.
The collection of the 64 codons makes up
the genetic code.
The genetic code was deciphered in 1966
by Nirenberg et al.
In the genetic code 61 codons specify 20
amino acids.
They are sense codons.
There are 3 codons, UAA, UAG and UGA,
which do not specify any amino acids.
They are stop codons ( termination codons,
nonsense codons ).
1.
Five Features of the Genetic Code
Universal
The genetic code is used by all species,
from prokaryotes to human being.
However some deviations are known to
occur in mitochondria and some unicellular organisms.
2. Directional
The sequence of triple-nucleotide codons is
read in the direction of 5’ to 3’.
The first codon of coding region of mRNA is
almost always AUG, the initiation codon.
The last codon of the coding region of mRNA
is one of the stop codons.
3. commaless
The coding region in mRNA is read in a
continuing way without punctuation.
If there is insertion or deletion of one or two
nucleotide(s) in the coding region of mRNA
frameshift mutation occurs.
AUG UCG CAA GAU ACG UCC
Met Ser Gln Asp Thr Ser
AUG CGC AAG AUA CGU CC
Met Arg Lys Ile Arg
AUG CUC GCA AGA UAC GUC C
Met Leu Ala Arg Tyr Val
4. degeneracy
Because 61 codons specify 20 amino acids
multiple codons must decode the same amino acid.
That is called degeneracy.
For example Leu, Ser, and Arg each is specified by
six different codons.
Homework
Find out other examples of codon degeneracy
5.
wobble
There is wobble in the process of codon
( in mRNA ) and anticodon ( in tRNA )
base-pairing.
That is the base-pairing does not strictly
according to the standard base-pairing
rule ( A-U , G-C , T-A ).
The wobble base pairs includes G-U, I-A,
I-C, and I-U, and often appear in the third
codon-anticodon position.
1,2, 3
1,2 3
codon CAU/C
AU A/C/U
anticodon GU G
UC I
The wobble base-pairing accounts for codon
degeneracy.
B
tRNA is the tool of transferring amino
acid.
There are 20 amino acids.
There are 61 sense codons.
There are about three dozens of prokaryotic
and about 50 eukaryotic tRNAs
Because of wobble base-pairing caused
codon degeneracy 61 sense codons can
be red by less than 61 tRNAs
It is the aminoacyl-tRNA that participates in
the protein synthesis.
The aminoacyl-tRNA synthetase catalyzes
the linkage of an amino acid to its cognate
tRNA.
C.Ribosome is the location of protein synthesis
A ribosome is composed of two subunits, a
a large one and a small one.
Prokaryotic
S 16SrRNA+Ps
30S
L 23SrRNA
5SrRNA
+Ps
50S
Whole 70S
Eukaryotic
18SrRNA+Ps
40S
28SrRNA
5.8SrRNA
5SrRNA
+Ps
60S
80S
1.
2.
Function of Ribosome
To hold mRNA, aminoacyl-tRNA and
translation factors in right place for protein synthesis.
To catalyze certain chemical reactions
in the process of protein synthesis
D . Other components for protein synthesis
20 amino acids
dozens of tRNAs
aminoacyl-tRNA synthetases
translation factors ( initiation, elongation,
and release factors )
ATP and GTP
Mg2+
Section Two
Protein Synthesis Takes Place in Five Stages
A
activation of amino acid and synthesis of
aminoacyl-tRNA
tRNAs are joined to amino acids to become
aminoacyl-tRNA in a reaction called aminoacylation.
Special enzymes called aminoacyl-tRNA
synthetases carry out the joining reaction
which is extremely specific.
nomenclature of tRNA and aminoacyl-tRNA
a.a. cognate tRNA c. synthetase aminoacyl-tRNA
ser
ser
Ser tRNA ser-tRNA synthetase ser-tRNA
leu
leu
Leu tRNA leu-tRNA synthetase leu-tRNA
The aminoacylation reaction is a two-step
reaction driven by ATP.
The first step is activation of amino acid.
AA + ATP + E ------------- AA-AMP-E + PPi
The second step is charging tRNA.
tRNA + AA-AMP-E ---- AA-tRNA+AMP+E
A cognate tRNA becomes attached to the
aminoacyl group through an ester bond.
The ester bond is formed between the acyl
group of the amino acid residue and the
tRNA’s 3’-OH.
Aminoacyl-tRNA synthetase has proofreading activity.
B
Formation of Initiation Complex
( in prokaryotes )
Initiation is the assembly of a ribosome with
fmet
fMet-tRNA
on an mRNA molecule.
How?
1.
IF1 and IF3 bind to a free 30S subunit.
This helps to prevent a large subunit
binding to it without an mRNA and forming an inactive ribosome.
2. mRNA binds to 30 S subunit by way of
mRNA and 16SrRNA interaction.
There is SD sequence ( RBS , ribosome
binding site ) 8-13 nt upstream of the
initiation codon in prokaryotic mRNA
which base-pairs with a complementary
sequence near the 3’ end of 16SrRNA.
5’ AGGAGGU 3’ SD sequence
3’ UCCUCCA 5’ on 16SrRNA
That results in the initiation codon in the
P site.
3. With the help of IF2 and GTP the initiator
fmet
tRNA ( fMet-tRNA
)can then bind to the
initiation codon ( AUG ) on the mRNA.
4. The 50S subunit can now bind, which
displaces IF1, IF2 and IF3, and GTP is
hydrolyzed to GDP and Pi.
The 70S initiation complex is formed.
met
fmet
There are two types of tRNA s , tRNA and
met
tRNA .
Both of them can link a Met, and form
fmet
met
Met-tRNA and Met-tRNA respectively.
fmet
The Met residue in Met-tRNA is formylated
fmet
and fMet-tRNA is formed.
fMet
fMet-tRNA is the initiator tRNA .
It recognizes only the initiation codon and
participates in translation initiation.
met
The Met residue in Met-tRNA will not be
modified.
It participates in translation elongation.
Eukaryotic Translation Initiation
met
There are also two types of tRNA s in eumet
met
karyotes, tRNAi and tRNAe .
met
Both of them can link Met and form Met-tRNAi
met
and Met-tRNAe .
met
Met-tRNAi is the initiator tRNA in eukaryotes.
met
Met-tRNAe participates in translation elongation.
Many eukaryotic initiation factors ( eIFs )
are required in eukaryotic initiation.
Cap and poly A tail participate in the initiation.
met
Met-tRNAi binds to 40S subunit before mRNA
binding
Homework
Read eukaryote translation initiation on P.377
Draw a comparison between prokaryotic and
eukaryotic translation initiation.
C
Translation Elongation
( in prokaryotes )
Elongation is a process of repeated ribosomal
cycles of amino acid addition.
With the formation of the 70S initiation complex
elongation begins.
The ribosomal cycle can be divided into three
steps, entrance ( registration ), peptide bond
formation and translocation.
When one ribosomal cycle is completed, the
nascent peptide is an amino acid residue
longer.
There is another extended sense of ribosomal
cycle, which is the cycle of translation.
It also has three stages, initiation, elongation
and termination.
When such a ribosomal cycle is completed, one
polypeptide is synthesized.
The Process of Elongation Cycle
1.
Entrance
Entrance begins when the P site is occupied by the initiator tRNA.
The complex of an elongation factor Tu
and GTP ( EFTu-GTP ) is required to deliver the aminoacyl-tRNA to the A site according to the codon-anticodon base-pairing
The energy is consumed in this step by
hydrolysis of GTP catalyzed by EFTu.
The EFTu-GDP is released.
Another elongation factor EFTs interacts
with EFTu and displaces GDP.
EFTu-GDP + EFTs-----EFTuTs + GDP
The EFTu-GTP complex is regenerated when
GTP displaces EFTs.
EFTuTs + GTP-------------EFTu-GTP + EFTs
Thus EFTu has GTPase activity and EFTs
is a GTP/GDP exchange protein.
2. Peptide Bond Formation
After aminoacyl-tRNA delivery the A and P
sites are both occupied.
The two amino acids ( fMet and AA ) are linked by
forming a peptide bond.
The 23SrRNA ( in eukaryotic ribosome the
28SrRNA ) catalyzes the peptide bond
formation between the acyl group of the
fMet residue and the alpha amino group
of the next AA residue.
So, protein biosynthesis is directional, from
N-terminal to C-terminal.
3. Translocation
A complex of elongation factor G and GTP
( EFG-GTP ) binds to the ribosome and in
an energy-consuming step.
The discharged tRNA from P site goes to
E site and then exits.
The peptidyl-tRNA is moved from the A site
to P site and the mRNA moved by one codon
relative to the ribosome.
EFG and GDP are released, the former being
reused.
A new codon is now present in the vacant A
site.
The elongation cycle is repeated until one
of the stop codons ( UAA, UAG,UGA )
appears in the A site.
D . Termination
Termination is the process of release of the
newly synthesized polypeptide.
Protein factors called release factors interact with stop codons and cause release of
the newly synthesized polypeptide.
There are three RFs in prokaryotes.
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 make 23SrRNA transfer
the polypeptide to water.
This reaction is driven by GTP and RF3 has
GTPase activity.
Thus the polypeptide is released from the
ribosome.
IF1 binds to the 70S ribosome.
The two subunits of the 70S ribosome then
dissociate. They participate in a new round
of translation initiation.
There is a single release factor in eukaryotes
the eRF.
It performs the roles carried out by RF1, RF2
and RF3 in prokaryotes.
When a ribosome has begun translating an
mRNA molecule and has moved about 70
- 80 nt from the initiation codon, a second
ribosome can assemble on the mRNA and
start translation.
When this second ribosome has moved along
a third can begin and so on.
Multiple ribosomes on a single mRNA are
called polysomes.
In this way protein synthesis performs with
high efficiency.
Section Three
Protein Folding and Posttranslational Processing
The newly synthesized polypeptide must
undergo folding and posttranslational processing so that it becomes the functional
protein with natural conformation.
A
Posttranslational Processing of
Newly Synthesized Polypeptide
1.N-terminal processing includes removing
N-formyl group, or N-fMet, ( in prokaryotes )
or N-Met ( in eukaryotes ), or several amino
acid residues at the N-terminal ( in both prokaryotes and eukaryotes ).
In eukaryotes N-terminal signal sequence
( signal peptide ) of membrane or secretory
proteins are removed.
There may be acetylation of N-terminal amino
acid residue.
C-terminal processing may also occurs.
2. Proteolytic Processing and Protein Splicing
Some proteins may undergo proteolytic processing.
The well-known example is the proteolytic processing of polyprotein POMC.
POMC is cleaved into different peptide hormones in different tissures.
It has been found that certain proteins in
prokaryotes have protein ‘ intron’.
The protein ‘ intron’ catalyzes self-splicing.
3. There are a lot of amino acid residue
modification patterns :
a. methylation
b. acetylation
c. phosphorylation
d. glycosylation
e. hydroxylation
f. disulfide bond formation
g. farnesylation
h. covalent binding of the prosthetic group
and so on
B
Protein Folding
As each nascent polypeptide emerges from
the ribosome, it begins to fold into its final
three-dimensional structure.
1.
Two Models of Protein Folding:
a. hierarchical folding model
secondary structuresuper-secondary
structure tertiary structure
b. molten globule model
hydrophobic interactionmolten globule
2. Molecular Chaperones Participate in
Protein Folding
Molecular chaperones are a kind of proteins
which help proteins to fold.
They have following functions
a. binding to the hydrophobic areas of the
unfolded protein thus preventing them
from abnormal aggregation
b . establishing an isolated environment
for protein folding
c. promoting protein folding and de-polymerization
d. in case of stress, unfolding the folded
protein
Molecular chaperones are divided into two
groups.
a. molecular chaperones which bind to ribosome such as TF, NAC.
b. molecular chaperones which do not bind
to ribosome such as Hsp families,PDI,and
PPI.
Molecular chaperones’ work is driven by ATP.
Homework
Read the text on pp.383, 384,and 385.
Summary the contents on these pages in
several sentences.
for example
Hsp70
1.The structure and function of Hsp70
2. Two helpers of Hsp70
3. The function of Hsp40
4. The function of GrpE
Tell the story of E coli GroEL in five sentences.
C
Polymerization of Subunits,
Multi-subunit Protein Formation
HbA formation
Free alpha peptide binds to nascent beta
peptide and alpha-beta dimer forms.
The dimer binds to two heme molecules.
Two heme-containing alpha-beta dimers
interact each other and form HbA.
Section Four
Clinical Relatives in Protein Synthesis
Protein synthesis is a regulated process.
The protein synthesis machinery responds
the environmental stimulation.
The synthesis of ferritin, a ferrous ion (Fe2+)
binding protein, is regulated by Fe2+.
Fe2+ binds to the 5’UTR of the ferritin mRNA
and stimulates ferritin synthesis.
A
Many viruses co-opt the host cell protein
synthesis machinery.
1.Virus mRNA is more efficiently translated
than host cell mRNA.
2. Viruses make abundant mRNA.
3.Some viruses can inhibit host cell mRNA
binding to 40S subunit.
B
Many antibiotics work because they selectively inhibit protein synthesis in bacteria.
Chloramphenicol inhibits prokaryotic peptidyl
transferase.
Streptomycin binding to 30S subunit causes
mRNA misreading.
Tetracycline binding to 30S subunit interferes
with aminoacyl-tRNA binding.
tyr
Puromycin is an analog of Tyr-tRNA and can
enter the A site during protein synthesis.
If puromycin occupies A site, translation elongation stops.
Puromycin was used in the study on protein
synthesis.
Cycloheximide inhibits eukaryotic peptidyl
transferase.
It is used in studies on protein metabolism.
Some bioactive substances that can inhibit
cell or virus protein synthesis
diphtheria toxin
Diphtheria toxin is a kind of enzyme.
It transfers ADP-ribose group from NAD+
to the eukaryotic elongation factor 2 (EF2).
That results in inactivation of EF2 and translation stops.
interferon
When mammalian cell is infected by virus,
the host cell can synthesize interferon.
Interferon has two ways to combat virus.
1
It induces synthesis of HCI, a kind of protein
kinase.
HCI catalyzes phosphorylation of eIF2.
The phosphorylated eIF2 is inactive.
Protein synthesis stops
2
Interferon induces an enzyme for the synthesis
of 2’5’oligoA.
2’5’oligoA is synthesized in the cell.
2’5’oligoA activates RNase L
RNase L hydrolyzes virus mRNA.