18,The anticodon and/or the amino acid
arms of a tRNA are key for a specific
aminoacyl-tRNA synthetase to recognize
? This was revealed via,crystal structure
determination of the synthetases complexed with
their cognate tRNAs; sequence comparison of
tRNAs binding to the same synthetase; and
mutagenesis studies.
? When the anticodon of tRNAVal is changed from
UAC to CAU,the mutated tRNAVal can be
recognized by Met-tRNA synthetase and generating
a Met-tRNAVal.
? When the 3:70 base pair on the amino acid arm of
tRNACys is changed from from C.G to G.U,the
mutated tRNACys is able to carry Ala,instead of Cys.
? A,microhelix” containing only 24 of the 76
nucleotides of tRNAAla is recognized and
aminoacylated by Ala-tRNA synthetase.
Positions (green and
orange) of
nucleotides involved
in recognition
between tRNAs and
aminoacyl-tRNA
synthetases.
A single G=U base pair is the only
element needed for specific binding
of tRNAAla and aminoacylation by
Ala-tRNA synthetase
19,Proofreading by some aminoacyl-
tRNA synthetases increases the fidelity
of protein synthesis
? The identity of the amino acid attached to a specific
tRNA is not checked by the ribosome (Ala-tRNACys
experiment).
? The calculated rate of incorrect incorporation of Val in
place of Ile is 1 in 200,but the observed is only 1 in
3000.
? Some synthetases are able to proofread the incorrectly
incorporated similar amino acids (e.g.,between Val and
Ile) at the aminoacyl-AMP stage or the aminoacyl-
tRNA stage.
? There seems to be separate proofreading active site
(s) on such synthetases.
? In a few synthetases that activate amino acids
having no close structural relatives,no such
proofreading activities have yet identified (selected
at the substrate binding level).
The codon on mRNA is recognized by the anticodon
of tRNA rather than by the activated amino acid
1,Cys-tRNACys is chemically converted to Ala-tRNACys ;
2,Studies with in vitro protein synthesis systems proved
that Ala-tRNACys will incorporate Ala at places of Cys:
using poly(G,U) as templates or hemoglobin mRNA as
template (with [14C]-Ala-tRNACys).
At the aminoacyl-
AMP stage
At the aminoacyl-
tRNA stage
Correctly
charged tRNA
Some synthetases are able to
proofread the incorrectly
incorporated amino acids
20,Met-tRNAiMet recognizes the
initiating AUG codon in almost all cells
? It was observed that about half of the N-terminal residues
of proteins in E.coli are Met,and the N-terminal of
nascent polypeptides is usually modified.
? There are two tRNAs for recognizing AUG and Met in all
organisms,one (tRNAiMet) brings Met to the initiating
AUG and the other (tRNAMet) brings Met to internal
AUGs.
? The Met charged on tRNAiMet (by Met-tRNA synthetase)
is specifically formylated by a transformylase to form
fMet-tRNAfMet in bacteria (Met-tRNAMet can not be
formylated).
? fMet-tRNAfMet can only enter and only fMet-tRNAfMet
can enter the initiation site (site P) on the ribosome in
E.coli.
Two types of methionine tRNA (tRNAiMet
or tRNAfMet in bacteria,and tRNAMet)
are found in all cells.
Both tRNAs are recognized by the same
Met-tRNA synthetase.
The Met charged on tRNAfMet
(by Met-tRNA synthetase) is
specifically formylated by
the action of transformylase
to form fMet-tRNAfMet
in bacteria;
N10-Formyltetrahydrofolate
provides the formyl group;
21,The Shine-Dalgarno sequence and a
nearby AUG marks the start site of
translation on a bacterial mRNA
? Sequence comparison of the initiator region of all
bacterial mRNAs revealed that a purine-rich
sequence called Shine-Dalgarno sequence is located
about 10 nucleotides upstream the initiating AUG
(or rarely GUG in bacteria) codon;
? This purine-rich sequence was found to be
complementary to a pyrimidine-rich sequence at the
3`end of the 16S rRNA present in the 30S subunit of
the ribosomes.
? The Shine-Dalgarno sequence may thus serve as the
ribosome binding site on bacterial mRNAs.
A purine-rich Shine-Dalgarno sequence and a AUG
codon marks the start site of polypeptide synthesis
on bacterial mRNA molecules,
Translation start codon
RBS
22,Aided by initiation factors,the mRNA,
the fMet-tRNAfMet and the ribosome
assemble to form an initiation complex
? The 16S rRNA directs the 30S subunit of to the
initiation site of the mRNA (through base pairing).
? IF3 binds to the 30S subunit to prevent its premature
association with the 50S subunit.
? IF2 (with GTP bound) then helps the fMet-tRNAfMet
to enter the P (peptidyl) site on the 30S subunit
where the initiation codon is located (no other
aminoacyl-tRNA can enter the P site!).
? The joining of the 50S subunit releases all the
initiation factors and forms the initiation complex.
The initiating complex is assembled
from the small subunit of the
ribosome,the mRNA,the initiating
aminoacyl-tRNA (being
fMet-tRNAfMet in bacteria),and the
large subunit of the ribosome.
23,The polypeptide chain is elongated
via a repeating three-step reactions
? The next aminoacyl-tRNA is delivered to the A
(aminoacyl) site of the ribosome by EF-Tu-GTP (step
1).
? EF-Tu (being about 5% of the total bacterial protein) is
believed to protect the activated ester bond from being
hydrolyzed by water.
? The milliseconds taken for GTP hydrolysis on EF-Tu
and milliseconds for EF-Tu-GDP to leave is believed to
allow the anticodon-codon interaction to be proofread.
? The fMet group is transferred to the second aminoacyl-
tRNA in the A site to form a peptide bond,generating a
dipeptidyl-tRNA (step 2).
? The 23S rRNA of the large subunit of the ribosome
seems to have the peptidyl transferase activity,
thus being a ribozyme (Noller,1992).
? EF-G (the translocase) then promotes the
translocation of the ribosome along the mRNA by
the distance of one codon,the deacylated tRNAfMet
is released from the E (exit) site of the ribosome,the
dipeptidyl-tRNA is relocated to the P site,and the A
site is open for the incoming (the third) aminoacyl-
tRNA (step 3).
AA2
Polypeptide
chain is
elongated on
the ribosome
Step 1:
Aminoacyl-
tRNA enters
the A site
(EF-Tu)
Step 2,peptide
bond formation
(23S rRNA)
Step 3:
Translocation
(EF-G)
The EF-Tu-GTP-
aminoacyl-tRNA
complexEF-Tu
Aminoacyl-tRNA EF-G hasa structure
similar to
EF-Tu-tRNA
24,Polypeptide synthesis is terminated
by release factors that read the stop
codons
? Two bacterial protein factors called RF1 and RF2
recognize the stop codons (RF1 for UAA and UAG;
RF2 for UAA and UGA),
? A third releasing factor RF3 is a GTP-binding protein
and seems to act in concert to promote the cleavage of
the peptide from peptidyl-tRNA.
? The specificity of the peptidyl transferase (23S rRNA)
is altered by the release factor,water become the
acceptor of the activated peptidyl moiety.
? A single eukaryotic release factor called eRF
recognizes all three stop codons.
The polypeptide chain is
released from the ribosome
when meeting a stop
codon (UAA,UGA,or
UGA)
25,At least four high-energy bonds are
ultimately consumed for the formation of
each specific peptide bond
? Two for activating each amino acid (ATP AMP +
2Pi).
? One for EF-Tu to deliver the aminoacyl-tRNA to the A
site of the ribosome (GTP GDP + Pi).
? One for EF-G to translocate the ribosome after each
peptide bond is formed (GTP GDP + Pi).
? The net free energy change ( ?G) in forming each
peptide bond between specific amino acids is about -
100 KJ/mol.
? This high energy consumption resulted in the nearly
perfect fidelity in translation.
26,Each mRNA is usually translated
simultaneously by many ribosomes (as
polysomes)
? This is observed in both prokaryotic and eukaryotic
cells.
? The guiding effectiveness of the short-lived mRNA
molecules is thus dramatically increased.
? In bacteria,the translation and transcription
processes are tightly coupled!
Polysomes are
regularly seen
in cells
In bacteria,translation
and transcription are
tightly coupled
27,The newly synthesized polypeptide
chains need to be folded,assembled,and
processed to make a functional protein
? Newly synthesized polypeptide chains will assume
their native conformation spontaneously or with
help from other proteins (called molecular
chaperones),with or without further
posttranslational modifications.
? The N-terminal formyl,fMet or Met may be
removed enzymatically.
? The N-terminal amino and C-terminal carboxyl
groups may be modified (e.g.,acetylated).
? Signal sequences (usually 15 to 30 residues in
length) at the N-terminal of some proteins will be
removed by specific peptidases after the protein
reached their cellular destinations (protein targeting),
? Specific amino acids in a protein may be
enzymatically modified posttranslationally by
groups like phosphate,carboxylate or methylate.
? Oligosaccharide groups are covalently attached to
specific Asn,Thr,or Ser residues in some proteins
(glycoproteins,often function extracellularly).
? Isoprenyl groups,which help anchor some proteins
in a membrane,may be added to some eukaryotic
proteins via a thioether bond (e.g.,G proteins,
lamins,and the ras protein).
? Addition of cofactors via covalent or noncovalent
bonds.
? Proteolytic cleavage of larger precursor proteins.
? Formation of disulfide bonds.
28,Nearly every step in protein synthesis
can be specifically inhibited by one
antibiotic or another
? Puromycin causes premature termination of
polypeptide synthesis by mimicking aminoacyl
transfer RNA,it is able to enter the A site and accept
the peptidyl group to form peptidyl puromycin,
which however will dissociate from the ribosome (in
both prokaryotes and eukaryotes).
? The concept of A and P sites arose from the use of
puromycin to ascertain the location of peptidyl-
tRNA,when peptidyl-tRNA is anchored in the A
site of the 30S subunit it can not react with
puromycin;
? Tetracyclin blocks the site A of bacterial ribosomes.
? Chloramphenicol inhibits the peptidyl transferase
activity of the 50S ribosomal subunit by binding to the
loop in 23S rRNA that interacts with the CCA sequence
of tRNA.
? Cycloheximide inhibits the peptidyl transferase activity
of the 60S ribosomal subunit (eukaryotes).
? Streptomycin causes misreading of genetic codes at low
concentrations and blocks fMet-tRNAfMet to enter the P
site at high concentrations (probably acts on the 16S
rRNA).
? Diphtheria toxin catalyzes the ADP-ribosylation of a
modified His residue on eEF2 blocking the translocation
of the peptidyl-tRNA from site A to site P.
? Ricin inactivates the 60S sununit of eukaryotic ribosomes.
Puromycin binds to
site A of ribosome,
accepts the peptidyl
group and then
leaves the ribosome.
29,Proteins synthesized from ribosomes
have to be targeted to specific cellular
destinations
? The synthesis of all proteins begins on free ribosomes
in the cytosol.
? A newly synthesized protein in E.coli can stay in the
cytosol,or be sent to the plasma membrane,the outer
membrane,the space between them,or the
extracellular medium.
? A newly synthesized protein in eukaryotic cells may
be targeted for secretion,integration into plasma
membrane,inclusion into specific organelles (e.g.,
nucleus,mitochondria,chloroplasts,lysosomes,
peroxisomes) or the cytosol.
30,The signal hypothesis for protein
targeting was proposed and proved
correct
? In the first version of the,signal hypothesis”,
Gunter Blobel postulated (in 1971) that proteins
secreted out of the cell contain an intrinsic signal
that governs them to and across membranes,
? In the more general version of the,signal
hypothesis”,he postulated (in 1980) each protein
carries in its structure the information needed to
specify its proper location in the cell.
? A range of signals that govern proteins to the
different parts of the cell have now been identified.
Proteins have
to be targeted
to various
cellular
locations
31,Signal sequences were revealed for
proteins targeting into the ER lumen
? Most lysosomal,membrane and secretary proteins
have a N-terminal signal sequence that directs them
for translocation into the lumen of the ER.
? These signal sequences ranging in length from 13 to
36 residues have the following features,a highly
hydrophobic stretch of 10 to 15 residues forms the
center of the signal,one or more positively charged
amino acid residues precedes the hydrophobic patch;
a small and neutral residue (being often Ala or Gly)
is located at the N-terminal side of the cleavage site.
Proteins are translocated to the ER lumen
via the guidance of their N-terminal signal
sequences,which share three similar
structural features.
32,Appropriate signal sequences and the
signal recognition particles (SRPs) direct
the ribosomes to the ER membrane
? The signal sequence of a nascent polypeptide chain
will be recognized by a SRP,a complex containing
one RNA (called the 7SL-RNA) and six different
proteins.
? The binding of SRP will halt polypeptide chain
elongation and direct the ribosome to ER membrane
via specific SRP and ribosome receptors.
? The nascent polypeptide chain is delivered to the
peptide translocation complex on the ER membrane.
? SRP then dissociates and chain elongation resumes.
? The signal sequence is then cleaved by a specific
peptidase in the ER lumen.
? The ribosome dissociates from the ER membrane
after the complete polypeptide chain is synthesized
and translocated into the ER lumen.
? The newly synthesized polypeptide chain is
processed and folded in the ER lumen.
A signal sequence directs the translocation of a polypeptide
chain to and cross the ER membrane.
33,Glycosylation plays a key role in
targeting certain proteins to lysosomes
? A 14-residue core oligosaccharide is built up by
sequential addition of monosaccharide units to a
dolichol-phosphate,which occurs on the cytosolic
and lumen surfaces of the ER membrane.
? The core is then enzymatically transferred en bloc to
the side chain of a specific Asn residue (in Asn-X-
Ser/Thr)of a growing polypeptide chain inside the
lumen of ER (cytosolic proteins are not glycosylated
as a result).
? Such glycosylated proteins and other proteins are
transported from ER to Golgi complex.
? O-linked oligosaccharides are added and N-linked
ones are further modified in the Golgi complex,
retaining a pentasaccharide core attached to Asn.
? Proteins are finally sorted for various destinations on
the basis of structural features,cell exterior,plasma
membrane or lysosomes.
? The presence of one or more mannose-6-P in the N-
linked oligosaccharides targets the hydrolases
specifically to lysosomes (Man is phosphorylated
using UDP-GlcNAc).
? Hydrolases would be secreted,instead of targeted to
lysosomes if cells are treated with tunicamycin,
which blocks the first step of the formation of the N-
linked oligosaccharides (glycosylation is essential for
targeting proteins to lysosomes).
An UDP-GlcNAc analog
34,Signals for targeting proteins to other
cellular locations are being identified
? Proteins targeted to mitochondria and chloroplasts
have a uptake-targeting sequence at the N-terminal
that will be cleaved once the proteins reached their
final destinations.
? Molecular chaperones are required for keeping
proteins in a partially unfolded form in cytosol and for
protein refolding in mitochondria and chloroplasts.
? Proteins targeted to nucleus have an internal signal
sequence that will not be cleaved after proteins reach
the nucleus.
? Proteins with a C-terminal KDEL are returned to the
ER.
35,Bacteria also use signal sequences to
target proteins
? The signal sequences for targeting proteins to the
inner and outer membranes,periplasmic space and
extracellular medium are much like those found on
eukaryotic proteins targeted to the ER (sometimes
replaceable).
? The proteins to be targeted to these locations are
maintained in a,translocation-competent”
conformation by Sec B (a molecular chaperone),
much like what happens to proteins to be targeted to
mitochondria and chloroplasts.
Bacteria also use signal sequences similar to
those for ER targeting to target proteins to
translocate outside the inner membrane.
36,Proteins differ markedly in their half
lives and are targeted for destruction
? Damaged proteins are usually quickly removed by
controlled degradation.
? Enzymes important in metabolic regulation usually
have short lives.
? Proteins are degraded by ATP-dependent cytosolic
systems in all cells.
? Ubiquitin,a extremely well conserved 76-residue
protein,tags proteins for destruction in eukaryotic
cells by the action of three enzymes (E1,E2 and E3).
? The free C-terminal Gly residue of ubiquitin is
ultimately linked to the amino group of the side
chain Lys residue of the target protein via an
isopeptide bond.
? The half-life of a cytosolic protein is determined to a
large extent by its amino-terminal residue.
? Other internal structural features (e.g.,the PEST
and RXXLGXIG sequences) have also been
identified for giving proteins short half lives.
? The ubiquinated proteins are finally degraded by
large multisubunit protease complexes called the
26S proteasomes,however leaving the ubiquitin
protein unaffected.
Summary
? Genetic and biochemical studies showed that the
genetic codes are continuous triplets or nucleotides.
? The genetic codes was deciphered within five years
using artificial mRNA templates of various base
composition (and triplets),in vitro protein synthesis
and filter binding assays.
? A tRNA molecule can recognize one to three codons
depending what the first (wobble) nucleotide of the
anticodon is (C and A for one,U and G for two,I for
three).
? In certain viral DNAs,overlapping genes are found.
? Protein synthesis occurs on ribosomes,having a
large and small subunits,both composing one or two
rRNA and many protein molecules.
? Protein synthesis can be divided into five stages,
activation of amino acids (ATP dependent,
aminoacyl-tRNA synthetase catalyzed); formation
of the initiation complex at the ribosome binding
site and initiation codon (helped by specific
initiation protein factors,IF-1,2,and 3); elongation
of the peptide chain (helped by the elongation
factors,EF-Tu,EF-Ts,EF-G and catalyzed by the
23S rRNA); termination (helped by the releasing
factors,RF1,RF2,and RF3); folding and
posttranslational modifications.
? Many antibiotics and toxins inhibit protein synthesis
at different steps.
? Proteins are targeted to various cellular locations
using signals intrinsic to the polypeptide chain or
glycosyl groups added posttranslationally and
helped by various molecular chaperones.
arms of a tRNA are key for a specific
aminoacyl-tRNA synthetase to recognize
? This was revealed via,crystal structure
determination of the synthetases complexed with
their cognate tRNAs; sequence comparison of
tRNAs binding to the same synthetase; and
mutagenesis studies.
? When the anticodon of tRNAVal is changed from
UAC to CAU,the mutated tRNAVal can be
recognized by Met-tRNA synthetase and generating
a Met-tRNAVal.
? When the 3:70 base pair on the amino acid arm of
tRNACys is changed from from C.G to G.U,the
mutated tRNACys is able to carry Ala,instead of Cys.
? A,microhelix” containing only 24 of the 76
nucleotides of tRNAAla is recognized and
aminoacylated by Ala-tRNA synthetase.
Positions (green and
orange) of
nucleotides involved
in recognition
between tRNAs and
aminoacyl-tRNA
synthetases.
A single G=U base pair is the only
element needed for specific binding
of tRNAAla and aminoacylation by
Ala-tRNA synthetase
19,Proofreading by some aminoacyl-
tRNA synthetases increases the fidelity
of protein synthesis
? The identity of the amino acid attached to a specific
tRNA is not checked by the ribosome (Ala-tRNACys
experiment).
? The calculated rate of incorrect incorporation of Val in
place of Ile is 1 in 200,but the observed is only 1 in
3000.
? Some synthetases are able to proofread the incorrectly
incorporated similar amino acids (e.g.,between Val and
Ile) at the aminoacyl-AMP stage or the aminoacyl-
tRNA stage.
? There seems to be separate proofreading active site
(s) on such synthetases.
? In a few synthetases that activate amino acids
having no close structural relatives,no such
proofreading activities have yet identified (selected
at the substrate binding level).
The codon on mRNA is recognized by the anticodon
of tRNA rather than by the activated amino acid
1,Cys-tRNACys is chemically converted to Ala-tRNACys ;
2,Studies with in vitro protein synthesis systems proved
that Ala-tRNACys will incorporate Ala at places of Cys:
using poly(G,U) as templates or hemoglobin mRNA as
template (with [14C]-Ala-tRNACys).
At the aminoacyl-
AMP stage
At the aminoacyl-
tRNA stage
Correctly
charged tRNA
Some synthetases are able to
proofread the incorrectly
incorporated amino acids
20,Met-tRNAiMet recognizes the
initiating AUG codon in almost all cells
? It was observed that about half of the N-terminal residues
of proteins in E.coli are Met,and the N-terminal of
nascent polypeptides is usually modified.
? There are two tRNAs for recognizing AUG and Met in all
organisms,one (tRNAiMet) brings Met to the initiating
AUG and the other (tRNAMet) brings Met to internal
AUGs.
? The Met charged on tRNAiMet (by Met-tRNA synthetase)
is specifically formylated by a transformylase to form
fMet-tRNAfMet in bacteria (Met-tRNAMet can not be
formylated).
? fMet-tRNAfMet can only enter and only fMet-tRNAfMet
can enter the initiation site (site P) on the ribosome in
E.coli.
Two types of methionine tRNA (tRNAiMet
or tRNAfMet in bacteria,and tRNAMet)
are found in all cells.
Both tRNAs are recognized by the same
Met-tRNA synthetase.
The Met charged on tRNAfMet
(by Met-tRNA synthetase) is
specifically formylated by
the action of transformylase
to form fMet-tRNAfMet
in bacteria;
N10-Formyltetrahydrofolate
provides the formyl group;
21,The Shine-Dalgarno sequence and a
nearby AUG marks the start site of
translation on a bacterial mRNA
? Sequence comparison of the initiator region of all
bacterial mRNAs revealed that a purine-rich
sequence called Shine-Dalgarno sequence is located
about 10 nucleotides upstream the initiating AUG
(or rarely GUG in bacteria) codon;
? This purine-rich sequence was found to be
complementary to a pyrimidine-rich sequence at the
3`end of the 16S rRNA present in the 30S subunit of
the ribosomes.
? The Shine-Dalgarno sequence may thus serve as the
ribosome binding site on bacterial mRNAs.
A purine-rich Shine-Dalgarno sequence and a AUG
codon marks the start site of polypeptide synthesis
on bacterial mRNA molecules,
Translation start codon
RBS
22,Aided by initiation factors,the mRNA,
the fMet-tRNAfMet and the ribosome
assemble to form an initiation complex
? The 16S rRNA directs the 30S subunit of to the
initiation site of the mRNA (through base pairing).
? IF3 binds to the 30S subunit to prevent its premature
association with the 50S subunit.
? IF2 (with GTP bound) then helps the fMet-tRNAfMet
to enter the P (peptidyl) site on the 30S subunit
where the initiation codon is located (no other
aminoacyl-tRNA can enter the P site!).
? The joining of the 50S subunit releases all the
initiation factors and forms the initiation complex.
The initiating complex is assembled
from the small subunit of the
ribosome,the mRNA,the initiating
aminoacyl-tRNA (being
fMet-tRNAfMet in bacteria),and the
large subunit of the ribosome.
23,The polypeptide chain is elongated
via a repeating three-step reactions
? The next aminoacyl-tRNA is delivered to the A
(aminoacyl) site of the ribosome by EF-Tu-GTP (step
1).
? EF-Tu (being about 5% of the total bacterial protein) is
believed to protect the activated ester bond from being
hydrolyzed by water.
? The milliseconds taken for GTP hydrolysis on EF-Tu
and milliseconds for EF-Tu-GDP to leave is believed to
allow the anticodon-codon interaction to be proofread.
? The fMet group is transferred to the second aminoacyl-
tRNA in the A site to form a peptide bond,generating a
dipeptidyl-tRNA (step 2).
? The 23S rRNA of the large subunit of the ribosome
seems to have the peptidyl transferase activity,
thus being a ribozyme (Noller,1992).
? EF-G (the translocase) then promotes the
translocation of the ribosome along the mRNA by
the distance of one codon,the deacylated tRNAfMet
is released from the E (exit) site of the ribosome,the
dipeptidyl-tRNA is relocated to the P site,and the A
site is open for the incoming (the third) aminoacyl-
tRNA (step 3).
AA2
Polypeptide
chain is
elongated on
the ribosome
Step 1:
Aminoacyl-
tRNA enters
the A site
(EF-Tu)
Step 2,peptide
bond formation
(23S rRNA)
Step 3:
Translocation
(EF-G)
The EF-Tu-GTP-
aminoacyl-tRNA
complexEF-Tu
Aminoacyl-tRNA EF-G hasa structure
similar to
EF-Tu-tRNA
24,Polypeptide synthesis is terminated
by release factors that read the stop
codons
? Two bacterial protein factors called RF1 and RF2
recognize the stop codons (RF1 for UAA and UAG;
RF2 for UAA and UGA),
? A third releasing factor RF3 is a GTP-binding protein
and seems to act in concert to promote the cleavage of
the peptide from peptidyl-tRNA.
? The specificity of the peptidyl transferase (23S rRNA)
is altered by the release factor,water become the
acceptor of the activated peptidyl moiety.
? A single eukaryotic release factor called eRF
recognizes all three stop codons.
The polypeptide chain is
released from the ribosome
when meeting a stop
codon (UAA,UGA,or
UGA)
25,At least four high-energy bonds are
ultimately consumed for the formation of
each specific peptide bond
? Two for activating each amino acid (ATP AMP +
2Pi).
? One for EF-Tu to deliver the aminoacyl-tRNA to the A
site of the ribosome (GTP GDP + Pi).
? One for EF-G to translocate the ribosome after each
peptide bond is formed (GTP GDP + Pi).
? The net free energy change ( ?G) in forming each
peptide bond between specific amino acids is about -
100 KJ/mol.
? This high energy consumption resulted in the nearly
perfect fidelity in translation.
26,Each mRNA is usually translated
simultaneously by many ribosomes (as
polysomes)
? This is observed in both prokaryotic and eukaryotic
cells.
? The guiding effectiveness of the short-lived mRNA
molecules is thus dramatically increased.
? In bacteria,the translation and transcription
processes are tightly coupled!
Polysomes are
regularly seen
in cells
In bacteria,translation
and transcription are
tightly coupled
27,The newly synthesized polypeptide
chains need to be folded,assembled,and
processed to make a functional protein
? Newly synthesized polypeptide chains will assume
their native conformation spontaneously or with
help from other proteins (called molecular
chaperones),with or without further
posttranslational modifications.
? The N-terminal formyl,fMet or Met may be
removed enzymatically.
? The N-terminal amino and C-terminal carboxyl
groups may be modified (e.g.,acetylated).
? Signal sequences (usually 15 to 30 residues in
length) at the N-terminal of some proteins will be
removed by specific peptidases after the protein
reached their cellular destinations (protein targeting),
? Specific amino acids in a protein may be
enzymatically modified posttranslationally by
groups like phosphate,carboxylate or methylate.
? Oligosaccharide groups are covalently attached to
specific Asn,Thr,or Ser residues in some proteins
(glycoproteins,often function extracellularly).
? Isoprenyl groups,which help anchor some proteins
in a membrane,may be added to some eukaryotic
proteins via a thioether bond (e.g.,G proteins,
lamins,and the ras protein).
? Addition of cofactors via covalent or noncovalent
bonds.
? Proteolytic cleavage of larger precursor proteins.
? Formation of disulfide bonds.
28,Nearly every step in protein synthesis
can be specifically inhibited by one
antibiotic or another
? Puromycin causes premature termination of
polypeptide synthesis by mimicking aminoacyl
transfer RNA,it is able to enter the A site and accept
the peptidyl group to form peptidyl puromycin,
which however will dissociate from the ribosome (in
both prokaryotes and eukaryotes).
? The concept of A and P sites arose from the use of
puromycin to ascertain the location of peptidyl-
tRNA,when peptidyl-tRNA is anchored in the A
site of the 30S subunit it can not react with
puromycin;
? Tetracyclin blocks the site A of bacterial ribosomes.
? Chloramphenicol inhibits the peptidyl transferase
activity of the 50S ribosomal subunit by binding to the
loop in 23S rRNA that interacts with the CCA sequence
of tRNA.
? Cycloheximide inhibits the peptidyl transferase activity
of the 60S ribosomal subunit (eukaryotes).
? Streptomycin causes misreading of genetic codes at low
concentrations and blocks fMet-tRNAfMet to enter the P
site at high concentrations (probably acts on the 16S
rRNA).
? Diphtheria toxin catalyzes the ADP-ribosylation of a
modified His residue on eEF2 blocking the translocation
of the peptidyl-tRNA from site A to site P.
? Ricin inactivates the 60S sununit of eukaryotic ribosomes.
Puromycin binds to
site A of ribosome,
accepts the peptidyl
group and then
leaves the ribosome.
29,Proteins synthesized from ribosomes
have to be targeted to specific cellular
destinations
? The synthesis of all proteins begins on free ribosomes
in the cytosol.
? A newly synthesized protein in E.coli can stay in the
cytosol,or be sent to the plasma membrane,the outer
membrane,the space between them,or the
extracellular medium.
? A newly synthesized protein in eukaryotic cells may
be targeted for secretion,integration into plasma
membrane,inclusion into specific organelles (e.g.,
nucleus,mitochondria,chloroplasts,lysosomes,
peroxisomes) or the cytosol.
30,The signal hypothesis for protein
targeting was proposed and proved
correct
? In the first version of the,signal hypothesis”,
Gunter Blobel postulated (in 1971) that proteins
secreted out of the cell contain an intrinsic signal
that governs them to and across membranes,
? In the more general version of the,signal
hypothesis”,he postulated (in 1980) each protein
carries in its structure the information needed to
specify its proper location in the cell.
? A range of signals that govern proteins to the
different parts of the cell have now been identified.
Proteins have
to be targeted
to various
cellular
locations
31,Signal sequences were revealed for
proteins targeting into the ER lumen
? Most lysosomal,membrane and secretary proteins
have a N-terminal signal sequence that directs them
for translocation into the lumen of the ER.
? These signal sequences ranging in length from 13 to
36 residues have the following features,a highly
hydrophobic stretch of 10 to 15 residues forms the
center of the signal,one or more positively charged
amino acid residues precedes the hydrophobic patch;
a small and neutral residue (being often Ala or Gly)
is located at the N-terminal side of the cleavage site.
Proteins are translocated to the ER lumen
via the guidance of their N-terminal signal
sequences,which share three similar
structural features.
32,Appropriate signal sequences and the
signal recognition particles (SRPs) direct
the ribosomes to the ER membrane
? The signal sequence of a nascent polypeptide chain
will be recognized by a SRP,a complex containing
one RNA (called the 7SL-RNA) and six different
proteins.
? The binding of SRP will halt polypeptide chain
elongation and direct the ribosome to ER membrane
via specific SRP and ribosome receptors.
? The nascent polypeptide chain is delivered to the
peptide translocation complex on the ER membrane.
? SRP then dissociates and chain elongation resumes.
? The signal sequence is then cleaved by a specific
peptidase in the ER lumen.
? The ribosome dissociates from the ER membrane
after the complete polypeptide chain is synthesized
and translocated into the ER lumen.
? The newly synthesized polypeptide chain is
processed and folded in the ER lumen.
A signal sequence directs the translocation of a polypeptide
chain to and cross the ER membrane.
33,Glycosylation plays a key role in
targeting certain proteins to lysosomes
? A 14-residue core oligosaccharide is built up by
sequential addition of monosaccharide units to a
dolichol-phosphate,which occurs on the cytosolic
and lumen surfaces of the ER membrane.
? The core is then enzymatically transferred en bloc to
the side chain of a specific Asn residue (in Asn-X-
Ser/Thr)of a growing polypeptide chain inside the
lumen of ER (cytosolic proteins are not glycosylated
as a result).
? Such glycosylated proteins and other proteins are
transported from ER to Golgi complex.
? O-linked oligosaccharides are added and N-linked
ones are further modified in the Golgi complex,
retaining a pentasaccharide core attached to Asn.
? Proteins are finally sorted for various destinations on
the basis of structural features,cell exterior,plasma
membrane or lysosomes.
? The presence of one or more mannose-6-P in the N-
linked oligosaccharides targets the hydrolases
specifically to lysosomes (Man is phosphorylated
using UDP-GlcNAc).
? Hydrolases would be secreted,instead of targeted to
lysosomes if cells are treated with tunicamycin,
which blocks the first step of the formation of the N-
linked oligosaccharides (glycosylation is essential for
targeting proteins to lysosomes).
An UDP-GlcNAc analog
34,Signals for targeting proteins to other
cellular locations are being identified
? Proteins targeted to mitochondria and chloroplasts
have a uptake-targeting sequence at the N-terminal
that will be cleaved once the proteins reached their
final destinations.
? Molecular chaperones are required for keeping
proteins in a partially unfolded form in cytosol and for
protein refolding in mitochondria and chloroplasts.
? Proteins targeted to nucleus have an internal signal
sequence that will not be cleaved after proteins reach
the nucleus.
? Proteins with a C-terminal KDEL are returned to the
ER.
35,Bacteria also use signal sequences to
target proteins
? The signal sequences for targeting proteins to the
inner and outer membranes,periplasmic space and
extracellular medium are much like those found on
eukaryotic proteins targeted to the ER (sometimes
replaceable).
? The proteins to be targeted to these locations are
maintained in a,translocation-competent”
conformation by Sec B (a molecular chaperone),
much like what happens to proteins to be targeted to
mitochondria and chloroplasts.
Bacteria also use signal sequences similar to
those for ER targeting to target proteins to
translocate outside the inner membrane.
36,Proteins differ markedly in their half
lives and are targeted for destruction
? Damaged proteins are usually quickly removed by
controlled degradation.
? Enzymes important in metabolic regulation usually
have short lives.
? Proteins are degraded by ATP-dependent cytosolic
systems in all cells.
? Ubiquitin,a extremely well conserved 76-residue
protein,tags proteins for destruction in eukaryotic
cells by the action of three enzymes (E1,E2 and E3).
? The free C-terminal Gly residue of ubiquitin is
ultimately linked to the amino group of the side
chain Lys residue of the target protein via an
isopeptide bond.
? The half-life of a cytosolic protein is determined to a
large extent by its amino-terminal residue.
? Other internal structural features (e.g.,the PEST
and RXXLGXIG sequences) have also been
identified for giving proteins short half lives.
? The ubiquinated proteins are finally degraded by
large multisubunit protease complexes called the
26S proteasomes,however leaving the ubiquitin
protein unaffected.
Summary
? Genetic and biochemical studies showed that the
genetic codes are continuous triplets or nucleotides.
? The genetic codes was deciphered within five years
using artificial mRNA templates of various base
composition (and triplets),in vitro protein synthesis
and filter binding assays.
? A tRNA molecule can recognize one to three codons
depending what the first (wobble) nucleotide of the
anticodon is (C and A for one,U and G for two,I for
three).
? In certain viral DNAs,overlapping genes are found.
? Protein synthesis occurs on ribosomes,having a
large and small subunits,both composing one or two
rRNA and many protein molecules.
? Protein synthesis can be divided into five stages,
activation of amino acids (ATP dependent,
aminoacyl-tRNA synthetase catalyzed); formation
of the initiation complex at the ribosome binding
site and initiation codon (helped by specific
initiation protein factors,IF-1,2,and 3); elongation
of the peptide chain (helped by the elongation
factors,EF-Tu,EF-Ts,EF-G and catalyzed by the
23S rRNA); termination (helped by the releasing
factors,RF1,RF2,and RF3); folding and
posttranslational modifications.
? Many antibiotics and toxins inhibit protein synthesis
at different steps.
? Proteins are targeted to various cellular locations
using signals intrinsic to the polypeptide chain or
glycosyl groups added posttranslationally and
helped by various molecular chaperones.
