Download Chen-6-Translation

Biochemistry
Chen Yonggang
Zhejiang University Schools of
Medicine
Translation, making protein following
nucleic acid directions
Bodega Bay,
Sonoma County
Breakfast at The Tides, Bodega Bay
The process of using base pairing
language to create a protein is termed
Translation
• Any process requires:
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–
–
–
A mechanism
Ribosome
Information-directions mRNA
Raw materials
amino acids / tRNA
Energy
ATP
• Any process has stages:
– Beginning
– Middle
– End
Initiation
Elongation
Termination
Translation requires a Dictionary
• The dictionary of Translation is called the
Genetic Code [Table 6.1]
• Correlates mRNA with Protein
– 3 nucleotides = 1 amino acid
• 4 possible nts
43= 64
20 possible aa
• 3 nucleotides read 5’→3’ are called a codon
– Codes for 1 amino acid
The Genetic Code
The Genetic Code
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Triplet
Non-overlapping
Unpunctuated
Degenerate
Nearly universal
Start signals
Stop signals
made of codons
read sequentially
once started, set frame
> than one codon/AA
mitochondrial code
AUG[met]
UAG, UAA, UGA
Players in Translation
• Ribosome
the machinery
• mRNA
the information
• Aminoacyl-tRNA the translator!
– Amino Acids/tRNA
– ATP
Ribosomes are ribonucleoprotein
complexes table 6.7
PROCARYOTIC
70 S
30S
EUCARYOTIC
80 S
Small subunit
60S
50S
RNA 5S, 16S,
23S
PROTEINS 55
40S
Large Subunit
RNA 5S,
5.8S,18S,28S
PROTEINS 84
Ribosomes must be assembled
with an mRNA
• The initiation process requires protein
factors
• A mRNA must be recognized and reading
frame must be set
• Aminoacyl-tRNAs must be available
5’
3’
Since the Translator is the AminoacyltRNA, it must be important
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•
Cells have 30+ tRNAs
tRNAs are redundant for some amino acids
Cells have 20 Aminoacyl-tRNA Synthetases
Aminoacyl-tRNA synthetases recognize 1
amino acid and 1 or more tRNAs
• Aminoacylation is very precise
Aminoacyl-tRNA Synthetases are
critical to Translation
• 1 Aminoacyl-tRNA Synthetase recognizes 1
Amino Acid and binds it
• 1 Aminoacyl-tRNA Synthetase recognizes 1
or more tRNAs specific for 1 amino acid
• The aminoacyl-tRNA Synthetase catalyzes
a two step reaction which overall is
AAx + tRNAx + ATP
Page 239
AAx-tRNAx + AMP + PPi
The first step involves forming an
enzyme-bound aminoacyl adenylate
O
E + ATP + R CH C O
+
H3N
O
O
E
..
R CH C O P O CH2
+
A
O
O
H3N
+ PPi
OO
HH
The hydrolysis of the PPi makes the process irriversible
O
O O
O P O CH2
R CH C O P O CH2
+
+
A
A
O
O
O
O
HN
3
tRNA
..O O
HH
The second step transfers
the amino acid to the 3’OH
of the tRNA, retaining the
energy of the adenylate
O
AMP O
HH
O
O P O CH2
A
O
O
tRNA
O OH
C O
R CH
+
H3N
tRNAs fold into L-shaped structures
Figure 2.59
Functional Sites of tRNAs
Figure 2.58
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•
•
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•
CCAOH 3’ Acceptor Sequence
Amino acid acceptor stem
D stem and loop
Extra loop
Anticodon stem and loop
Anticodon
TyC stem and loop
5’ Terminus
The anticodon forms antiparallel base
pairs with a codon in the mRNA
• Each tRNA has a unique anticodon
• There are 61 codons which base pair with tRNA
anticodons, most pairing is Watson-Crick but
Wobble in the 5’ base of the anticodon allows
degeneracy
• 3 codons do not normally base pair with
anticodons-UAA, UAG, UGA. The lack of a
complementary anticodon-Termination Codons
Wobble allows one codon to base
pair with up to three anticodons
3'
5'
Base stacking in the
anticodon assures
that bases 2 and 3 of
the anticodon will
follow Watson-Crick
rules. Base 1 can
wobble
mRNA
GAG UGC GCU
ACG
3'
5'
tRNA
cys
Depending on base 1 it can pair
with 1,2 or 3 bases
• If the wobble base is U, it can H bond to A
(expected) or G (unexpected).
• If the wobble base is G, it can H bond to C
(expected) or U (unexpected).
• A and C form only the expected base pairs.
• Inosine in the wobble position can H bond
to A, C, and U.
Thus 31 tRNAs can read 61 codons
Translation takes place in three
stages
• Initiation-- once per protein it gets the
system in motion
• Elongation-- repeated for each codon in the
mRNA making a peptide bond
• Termination-- finishes and releases the
newly synthesized protein
Initiation
A common mechanism
Procaryotic initiation assembles the pretranslational complex
• Mechanism is similar for eucaryotes and
procaryotes [differences are important]
• Components:
– Small subunit containing a specific mRNA
sequence(Shine-Dalgarno) which guides the mRNA
into correct position for reading frame relative to the
16S rRNA
– Proteinaceous initiation factors
– Initiator AA-tRNA
– mRNA(monocistronic for eucaryotes, polycistronic for
procaryotes)
Differences in the process provide the
basis for specific antibiotic action
• Procaryotes
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30S ribosomal subunit
IF-1, IF-2, IF-3
fMet-tRNAMetF
GTP
• Eucaryotes
• 40S ribosomal subunit
• eIF-2a, eIF-3, eIF-4a, eIF4c, eIF-4e, eIF-4g, eIF-5,
eIF-6
• Met-tRNAMeti
• GTP
Initiation Factors have Specific Roles
• Procaryotes
• IF-3 binds 30S
• IF-2 binds initiator
AA-tRNA
• IF-1 GTP hydrolysis
• RNA:RNA base
pairing indexes
mRNA
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Eucaryotes
eIF-2 itRNA Binding
eIF-3 40S anti-association
eIF-4g binds mRNA
eIF-4e cap binding
eIF-4a mRNA indexing
eIF-4c ribosomal i AA-tRNA
eIF-5 GTP hydrolysis
eIF-6 60S anti-association
In procaryotes IFs 1,2 and 3 are
needed to begin
IF-3 is an 30S anti-association factor
IF-2 binds and preps initiator AA-tRNA
IF-1 is a GTP binding hydrolase
These allow the association of the 30S, MettRNA metF and factors to bind in preparation
for mRNA and 50S binding
Initiation is
similar for
pro- and
eucaryotes
Devlin 6.7
Intiation occurs once per translational
cycle
• The preinitiation complex is formed on the small
subunit
• GTP is bound to initiation factors. GTP hydrolysis
carries out a process and drives a conformational
change which leads to the next activity
• The mRNA is indexed to appropriate AUG codon
• The mRNA is locked into the cleft between small and
large subunits
• Addition of the large subunit creates A , P and E sites on
the ribosome
• The initiator AA-tRNA is locked into the P site
Eucaryotic
initiation is
similar
Devlin 6.7
Eucaryotic initiation has
differences
• The mRNA is not indexed by the ribosomal rRNA
(eukaryotic mRNAs do not have Shine-Dalgarno
sequence)
• Cap binding is essential for initiation
• The initiation complex does not use formylated
methionine but does use a specific initiator
Methionine-specific aminoacyl-tRNA for
initiation
• Protein synthesis occurs at the first AUG
The association of all initiation
components creates a 70S ribosome
with initiator tRNA in the P site
5'
3'
AUG
mRNA
CAU
UAC
E
fmetP
A
GCU
Elongation
A repeated experience
Once initiation is complete the ribosome
is ready for elongation
• Elongation is the process of addition of amino
acids to the C-terminus of the growing
polypeptide
• Synthesis of each peptide bond requires energy
derived from the cleavage of the AA-tRNA ester
bond. The ribosomal enzyme doing this is called
Peptidyl Transferase
• Elongation is repeated as many times as there are
codons in the mRNA
As is the case for initiator tRNA all
aminoacyl-RNAs must be present for
protein synthesis
• Good nutrition requires that all amino acids
must be available in the diet
• For procaryotes most can be synthesized at
an expense of energy
• Eucaryotes are able to form some but not all
amino acids, thus some are essential in the
diet
Pools of AA-tRNAs are formed by the
Aminoacyl-tRNA Synthetases
• AA-tRNA synthetases recognize 2o
and 3o structure near the TyC,D, and
extra loop and the acceptor stem on
the L-shaped tRNA molecules
• AA-tRNA synthetases recognize 3dimensional structure and functional
groups of the amino acids
• As we saw earlier, AA-tRNA
synthetases use ATP to form a highenergy ester bond at the 3’OH on the
tRNA
Once an AAx-tRNAx is formed, the Amino
Acid becomes Invisible
• The ribosome mediates the association
between codons on the mRNA and anticodons
on the tRNA
• Specificity of AA incorporation depends upon
the anticodon of the tRNA
• Whatever is on the tRNA will be incorporated
into the protein at the site
• The tRNA adapts the AA to the specified site
Following Initiation the Ribosome has 3
functional sites
• A site-aminoacyl-tRNA
binding site [incoming AAtRNA, only initiator AAtRNA goes to the P site]
• P site-peptidyl-tRNA
binding site[attachment of
growing polypeptide site
• E site-spent tRNA exit site
E
P
A
Each elongation cycle requires
elongation factors
• Procaryotes
• Eucaryotes
• EF-T AA-tRNA binding to A • EF-1 AA-tRNA binding to A
site, GTP binding/hydrolysis
site, GTP binding/hydrolysis
• EF-G GTP hydrolysis,
• EF-2 GTP hydrolysis, ribosomal
ribosomal conformational
conformational change, index
change, index peptidyl-tRNA to peptidyl-tRNA to P site,
P site, expulsion of spent tRNA
expulsion of tRNA from E site
from E site
In procaryotes, under the control
of EF-T, a second aminoacyltRNA is bound in the A site
5'
3'
AUG
UAC
mRNA
fmet
E
P
CAU
GUA
his
A
GCU
In eucaryotes
similar events
occur
Devlin 6.8
Hydrolysis of bound GTP changes the
conformation of the Ribosome
• The conformational change locks the
aminoacyl-tRNA into the A site
• Brings the anticodon in close approximation
with the codon
• Prepares the ribosome for binding of
another GTP binding hydrolase EF-G
The energy for peptide bond formation derives
from the aminoacyl-tRNA ester bond
• Cleaving the ester bond provides energy for the
formation of a peptide bond
• Catalysis is most likely provided by an integral 50/60S
ribozyme, the peptidyl transferase, an RNA-containing
enzyme(parts of the 23s rRNA) in the ribosome
• Upon synthesis of the peptide bond, the growing
polypeptide chain is linked to the tRNA on the P site
Peptidyl transferase synthesizes a
peptide bond forming a dipeptide
5'
3'
AUG
UAC
mRNA
CAU
GUA
his
E
P
A
fmet
GCU
The peptide bond is
formed using the
energy derived from
the aminoacyl ester
bond and moves the
peptide to the A sitebound AminoacyltRNA
Following peptide bond formation a new
factor drives translocation of the peptide
• Specificity provided by antiparallel codonanticodon pairing between A site-bound AAtRNA and mRNA
• Translocation driven by EF-G/2 catalyzed GTP
hydrolysis-derived conformational change
• mRNA ratchets 5’→3’ through the ribosome
moving the C(codon):AC(anticodon) from A to
P site by the action of a translocase
• Time to find AA-tRNA is important to fidelity
EF-G mediated GTP hydrolysis
translocates the mRNA and peptidyltRNA expelling the spent tRNA
5'
mRNA
3'
AUG
UAC
CAU
GUA
his
E
GCU
P
fmet
A
Eucaryotic
translocation
is similar
Devlin 6.8
This elongation cycle is repeated as
many times as there are codons
EF-T/1 mediated binding is followed
peptide bond formation and EF-G/2
mediated peptidyl transfer
Eucaryotic elongation is similar
to the procaryotic process
Repeat of 3 steps in elongation cycle
1. Binding of an incoming AA-tRNA
2. Peptide bond formation, catalyzed by
peptidyl transferase
3. translocation, done by translocase
The growing
polypeptide
chain remains
attached to the
last tRNA added
The next codon
is UAG
When a termination codon occupies the the A
site no AA-tRNA will bind
• Termination codons work because no tRNA has a
complementary anticodon
• When the site is occupied by UAA, UAG or UGA time
passes without A site occupancy by an AA-tRNA
• This allows binding of release or termination factors,
proteins[size and shape of tRNAs] that change the activity
of peptidyl transferase to a peptidyl hydrolase and thus
mediate release of the polypeptide from the ribosome
Termination requires proteinaceous
termination factors
• Procaryotes
• Eucaryotes
• Release Factor GTP
• eRF GTP binding, GTP
binding, GTP hydrolysis,
hydrolysis, conformational
conformational change,
change, cleavage of 3’cleavage of 3’-peptidylpeptidyl-CCAOH ester
CCAOH ester linkage,
linkage, expulsion of
expulsion of polypeptide,
polypeptide, dissociation
dissociation of 30S and
50S subunits
of 40S and 60S subunits
Devlin 6.10
Polysome
In both prokaryotes and eukaryotes,
mRNAs are read simultaneously by
numerous ribosomes, An mRNA with
several ribosomes bound to it is referred to
as a polysome.
Posttranslational modification
• Some newly made proteins, both
prokaryotic and eukaryotic, do not attain
their final biologically active conformation
until they have been altered by one or more
processing reactions called posttranslational
modification
Different ways of modification
• Amino-Terminal and Carboxyl-Terminal
Modification
• Loss of Signal Sequence: the 15 to 30 residues at
the amino-terminal end of some proteins play a
role in directing the protein to its ultimate
destination in the cell. Such signal sequences are
ultimately removed by peptidase
• Modification of Individual Amino Acids:
The hydroxyl groups of Ser, Thr, and Tyr can be
phosphorylated , some others can be carboxylated
and methylated.
Different ways of modification
• Attachment of Carbohydrate Side Chains: such as
glycoproteins, N-linked oligosaccharides (e.g.
Asn), O-linked-oligosaccharides(e.g. Ser or Thr)
• Addition of Isoprenyl Groups
• Addition of Prosthetic Groups:Two examples are
the biotin molecule of acetyl-CoA carboxylase
and the heme group of hemoglobin or cytochrome
c.
Different ways of modification
• Proteolytic Processing: proinsulin and
proteases such as chymotrypsinogen and
trypsinogen(zymogen activation)
• Formation of Disulfide Cross-link:
intrachain or interchain disulfide bridges
between Cys residues
Because of differences in translation
bacterial growth can be inhibited by
antibiotics
Devlin 6.8
Eucaryotes can be targeted by
microorganisms
• Diphtheria toxin carries out its effects by
mediating a covalent modification of eEF-2
NAD++ EF-2
ADP-Ribose-EF2 + Nicotinamide
• ADP-ribosylated eEF-2 is ineffective, thus
interrupting polypeptide synthesis
What’s Next?
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Once made can proteins be modified?
How is protein folding effected?
How are proteins exported after synthesis?
How is protein turnover controlled?