Chapter 27 Protein Metabolism
1,A brief history of understanding protein
metabolism;
2,The studies leading to the deciphering of
the genetic codes;
3,The pathway leading to the synthesis of
a functional protein;
4,Current understanding on protein
targeting and degradation.
1,Translation (protein synthesis)
necessitates the coordinated interplay of
about 300 macromolecules in the cells
? The most complex of all biosynthetic pathways.
? 60 to 90 macromolecules for making up the protein-
synthesizing machine ribosomes
? Over 20 enzymes for activating the amino acids.
? Over 10 auxiliary proteins for the initiation,elongation
and termination of the polypeptide chains.
? Account for up to 90% of the chemical energy used by a
cell for all biosynthetic reactions.
? The molecules used for translation account for
more than 35% of the cell’s dry weight,
? However,proteins are synthesized with very high
efficiency,a complete polypeptide chain of 100
residues is synthesized in about 5 seconds in an
E.coli cells at 37oC.
2,The molecular mechanism of protein
synthesis was mainly revealed during the
2nd half of the 20th century
? Ribonucleoprotein particles (were later called ribosomes)
were revealed to be the site of protein synthesis in rat liver
cells,using radioactively labeled amino acids and
immediate subcellular fractionations (early 1950s,by
Zamecnik).
? Amino acids were found to be activated by attaching to a
special form of heat-stable RNA molecules (later called
tRNAs) before being incorporated into polypeptides (1950s,
by Hoagland and Zamecnik).
? Each tRNA molecule was found to function as an
adapter (originally hypothesized by Francis Crick),
carrying a specific amino acid with one site and
recognizing a specific site on a template with another site,
? The concept of messenger RNA (mRNA) was boldly
formulated by Jacob and Monod in 1961,a short-lived
RNA should serve as the information carrier between
gene and protein (to explain the quick induction of
proteins in E.coli).
? This bold hypothesis was quickly confirmed by studies
of E.coli cells infected by T2 phages,
Ribosomes were revealed to be the site of
protein synthesis in early 1950s (pulse
labeling with radioactive amino acids and
subcellular fractionations).
Crick’s
adapter
hypothesis
Hydrogen
bonds
3,Amino acids in a polypeptide chain
were found to be coded by groups of
three nucleotides in a mRNA
? Simple calculation indicated that three or more bases are
probably needed to specify one amino acid.
? Genetic studies of insertion,deletion,and substitution
mutants showed codons for amino acids are triplet of
nucleotides; codons do not overlap and there is no
punctuation between codons for successive amino acid
residues.
? The amino acid sequence of a polypeptide is defined by a
linear sequence of contiguous codons,the first codon
establishs a reading frame.
Genetic studies showed that genetic
codons are successive triplets of nucleotides
Altered amino acid sequences
Amino acid sequence studies of tobacco mosaic
virus mutants and abnormal hemoglobins
showed that alterations usually affected only
one single amino acid,genetic codes are
nonoverlapping.
Each mRNA molecule would have three
potential reading frames (but only one
usually codes for a polypeptide chain),
4,The genetic codes were deciphered by
simply using the in vitro protein synthesis
system
? Artificially synthesized poly(U),synthesized using
polynucleotide phosphorylase,was added to 20 reaction
tubes each containing the cell-free E.coli extract,GTP,
ATP,a mixture of 20 amino acids,and one 14C labeled
amino acid.
? Radioactive polypeptide was only detected in the tube
containing [14C]-Phe (with high concentration of Mg2+ ).
? When poly (A) and poly (C) were added,radioactive
polypeptides were only detected in the tubes containing
14C-labeled L-Lys and L-Pro respectively.
? UUU,AAA,CCC encodes Phe,Lys,Pro
respectively.
? When poly(G) was added,no polypeptides
synthesized prabably due to the formation of
tetraplexes of the poly (G) strands.
5,Base composition of the triplets coding
for some amino acids were revealed
using mixed copolymers of RNA
? The composition of an RNA synthesized using
polyribonucleotide phosphorylase depends on the
proportion of each NDP present in the reaction mixture.
? Investigation of the identity and quantity of the amino acids
incorporated into the polypeptides in response to random
polymers of RNA made from various ratios of NDPs can
reveal the nucleotide composition (but not exact sequence)
of the triplets corresponding to certain amino acids.
Composition of triplets coding for certain amino acids were assigned
6,Many trinucleotides were found to
promote the binding of specific
aminoacyl-tRNA to ribosomes
? It was discovered in 1964 that a specific aminoacyl-
tRNA would bind to the isolated ribosomes when
the corresponding synthetic polynucleotide
messenger or only the trinucleotide is present.
? Many genetic codes were revealed by examining
which aminoacyl-tRNA is bound to the ribosomes
mixed with specific trinucleotides using filter-
binding assay.
The filter-binding assay
for detecting the binding
of a trinucleotide to a
specific aminoacyl-tRNA
molecule,about 50
codons were assigned by
this simple and elegant
method.
7,Polyribonucleotides of defined
repeating sequences of two to four bases
helped to end the decoding work
? Khorana successfully developed a method to synthesize
polyribonucleotides of defined repeating sequences
using a combination of organic synthesis and enzymatic
techniques.
? Polypeptides synthesized using these
polyribonucleotides had repeating one to a few amino
acids.
? Sequences for specific genetic codes can be determined
by comparing the information obtained here and those
obtained by using RNAs having random sequences
made from two nucleotides of determined ratio.
Copolymer of repeating dinucleotides always
lead to synthesis of polypeptides of repeating
dipeptides:
ABABABABABABAB--
aa1---aa2---aa1---aa2----
Copolymer of repeating trinucleotides will lead
to the synthesis of three homopolypeptides:
ABCDABCDABCDABCDABCDABCDABCDABCD----
ABCDABCDABCDABCDABCDABCDABCDABCD----
ABCDABCDABCDABCDABCDABCDABCDABCD----
ABCDABCDABCDABCDABCDABCDABCDABCD----
Copolymer of repeating tetranucleotides will lead
to the synthesis of a single type of polypeptide with repeating
tetrapeptides.
Three different
homopolypeptides are
produced from most
polyribonucleotides
consisting of repeating
sequences of three
nucleotides;
one type of polypeptide
containing repeating
tetrapeptides was always
produced from
polyribonucleotides
consisting of repeating
sequences of four
nucleotides.
8,All 64 triplet codes were deciphered by
1966
? 61 of the codons code for the 20 amino acids and three
(UAA,UAG,UGA) for chain termination,called
termination codons,stop codons,or nonsense codons).
? AUG is a dual codon coding for initiation and Met.
? 18 of the amino acids are coded by more than one codon,
the genetic codes are degenerate.
? The codes seem to have evolved in such a way to minimize
the deleterious effects of mutations,especially at the third
bases,XYU and XYC always encode the same amino acid
XYA and XYG usually code for the same amino acid.
? A reading frame codes for more than 50
amino acids without a stop codon is called
an open reading frame,which has the
potential of encoding a protein.
All 64
genetic
codes
Established
the chemical
structure of
tRNA
Established the in
vitro system for
revealing the
genetic codes
Devised methods
to synthesize RNAs
with defined
sequences
9,The genetic code has been proved to
be nearly(not absolutely) universal
? Direct comparisons of the amino acid sequences of proteins
with the corresponding base sequence of their genes or
mRNAs,as well as recombinant DNA technologies,proved
that the genetic codes deciphered from in vitro studies were
correct and almost universally applicable.
? A small number of,unusual codes” have been revealed in
many mitochondria genomes and nuclear genome of a few
organisms,
10,Overlapping genes were found in
some viral DNAs
? Genes usually do not overlap.
? The 5.3 kb DNA of bacteriophage fX174 was found to be
not long enough to code for the ten proteins it produces.
? Detailed sequence correlation of the viral DNA and the
protein sequences revealed the,genes within genes”
phenomena.
? The overlapping genes use different reading frames.
? This phenomena was also found in other viruses (including
l phage,SV40).
Some genes
overlap in the
fX174
bacteriophage
DNA
11,Three kinds of RNA molecules
perform different but cooperative
functions in protein synthesis
? mRNAs carry the genetic information copied from DNA in
the form of genetic codons.
? tRNAs mediate the incorporation of specific amino acids
according to genetic codons present on the mRNA
molecules via their specific anticodon triplets.
? rRNAs associate with a set of proteins to form the protein-
synthesizing machines (ribosomes) and probably catalyze
peptide bond formation during protein synthesis.
Roles of the
3 types of
RNAs in
translation
All tRNAs have common structural features:
cloverleaf in secondary,“L” in 3-D structures.
12,Some tRNA molecules can recognize
more than one codons via wobble pairing
? The adapter tRNAs recognize the codons on a mRNA via a
triplet called anticodons.
? It was first proposed that a specific tRNA anticodon would
exist for every of the 61 (or 64) codons,but less tRNAs
were revealed.
? It was revealed that highly purified tRNA molecules (e.g.,
alanyl-tRNAAla) of known sequence could recognize several
different codons.
? Inosine,which may form base pair with A,U,and C,was
found to be present at the first position of the anticodons in
some tRNAs.
? Crick proposed the,wobble hypothesis” in 1966 to explain
the pairing features between anticodons and codons:
– The first two bases of a codon in mRNA confer most of
the coding specificity,the third base can be loosely
paired with the anticodons;
– The first base of some anticodons can wobble and
determines the number of codons a given tRNA can read
(A and C for one,U and G for two,I for three);
– Codons that specify the same amino acid but differ in
either of the first two bases need different tRNAs,i.e.,a
mininum of 31 tRNA are needed to translate the 61
codons;
? This hypothesis has been widely supported by all the
evidence gathered since (thus the,wobble rule”).
? This moderate pairing strength may serve to
optimize both the accuracy and speed of polypeptide
synthesis.
The codon-anticodon pairing between the a mRNA and a
tRNA,the presence of an inosinate residue at position one
in the anticodon allows the tRNA to recognize a few
codons.
I
I I
C
U
A
G U
Possible wobble pairing between anticodon and codon.
13,Ribosomes are the protein-
synthesizing machines
? All ribosomes consist of two units of unequal size.
? The large unit contain two or three rRNA molecules and 31
or 50 proteins.
? The small unit contain one rRNA molecule and 21 or 33
proteins.
? The total size of the prokaryotic and eukaryotic ribosomes
are 70S and 80S respectively.
? The rRNA and protein components of the bacterial
ribosomes have been separated and successfully
reconstituted in vitro.
Three rRNA
52 proteins
Four rRNA
83 proteins
Ribosomes are ribonucleoprotein particles for
synthesizing proteins.
Structure of
70S ribosome
at 5.5 A
50S
30S
14,Pulse-labeling (isotope tracer) studies
revealed that polypeptide synthesis
begins at the N-terminal
? 3H-leucine was added to reticulocyte cells actively
synthesizing hemoglobin for a short period of time.
? a and b chains of hemoglobin were isolated,treated with
trypsin and analyzed by fingerprinting and autoradiography.
? A gradient of radioactivity increasing from the amino to
carboxyl end of each chain was detected,indicating that the
carboxyl end was synthesized last,the polypeptide chain
grows by successive addition of amino acids at the C-
terminal.
Pulse-labeling (isotope tracer) studies revealed that
polypeptide synthesis begins at the N-terminal
15,mRNA is efficiently translated by
polysomes in the 5` 3` direction
? When the synthetic polynucleotide AAA(AAA)nAAC
was used as templates to guide polypeptide synthesis in
a cell-free protein-synthesizing system,the polypeptide
Lys-(Lys)n-Asn was produced.
? Translation undergoes from 5` to 3` along the mRNA.
? EM studies showed that multiple ribosomes (as
polysomes) can translate one single mRNA
simultaneously in all cells.
? Transcription and translation are closely coupled in
bacteria.
? Many eukaryotic polysomes are circular,which may
allow rapid recycling of ribosomes for translation.
Electron microscopic
examination of coupled
transcription and
translation in E.coli.
Direction of
transcription
A single mRNA is usually
translated by multiple
ribosomes (polysomes)
simultaneously.
Formation of circular eukaryotic mRNA by protein-
protein interactions of eIF4E and eIF4G (binding to
the m7G cap),poly(A)-binding protein I (PABI)
Force-field
electron micrograph
Model of protein synthesis on circular polysomes and
recycling of ribosomal subunits.
16,The synthesis of a protein can be
divided into five stages
? Each amino acid is first covalently attached to a specific
tRNA molecule in a reaction catalyzed by a specific
aminoacyl-tRNA synthetase (Stage 1).
? The mRNA then binds to the smaller subunit of the
ribosome,after which the initiating aminoacyl-tRNA and
the large subunits of the ribosome will bind in turn to form
the initiating complex (Stage 2)
? The first peptide bond is then formed after the second
aminoacyl-tRNA is recruited with help of the elongation
factors,and the chain is then further elongated (stage 3).
? When a stop codon (UAA,UAG,and UGA) is met,
the extension of the polypeptide chain will come to a
stop and is released from the ribosome with help
from release factors (Stage 4).
? The newly synthesized polypeptide chain has to be
folded and modified (in many cases) before
becoming a functional protein (Stage 5).
Each amino acid is
specifically attached
to a specific tRNA
before used for
protein synthesis
Stage 1
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.
Stage 2
AA2
Polypeptide chain
is elongated on
the ribosome
Stage 3
The polypeptide chain is
released from the ribosome
when meeting a stop
codon (UAA,UGA,or
UGA)
Stage 4
17,The 20 aminoacyl-tRNA synthetases
attach the 20 amino acids to one or more
specific tRNAs
? An amino acid is first activated to form an aminoacyl-AMP
intermediate (can be isolated when tRNA is absent),and is
then charged to one or more specific tRNAs all catalyzed by
one such specific aminoacyl-tRNA synthetase.
? The 20 synthetases have diverse sizes,subunit composition,
and amino acid sequences and are categorized into two
classes,class I and II,which bind to opposite faces of the
incoming tRNAs,link the amino acids to the 2`-OH and 3`-
OH groups of the terminal adenosine respectively.
Aminoacyl-tRNA synthetases
can be divided into two classes
based on differences in structure
and reaction mechanisms.
Aminoacyl-tRNA
synthetase
3 binding sites
in the active site
Aminoacyl-
AMP
Aminoacyl-
tRNA
Each synthetase
charges a
specific
tRNA with
a specific
amino acid
“the second
genetic codes”.
Gln-tRNA synthetase
(type I,monomeric)
Asp-tRNA synthetase
(type II,dimeric)
ATP
Amino acid arm
Anticodon arm
1,A brief history of understanding protein
metabolism;
2,The studies leading to the deciphering of
the genetic codes;
3,The pathway leading to the synthesis of
a functional protein;
4,Current understanding on protein
targeting and degradation.
1,Translation (protein synthesis)
necessitates the coordinated interplay of
about 300 macromolecules in the cells
? The most complex of all biosynthetic pathways.
? 60 to 90 macromolecules for making up the protein-
synthesizing machine ribosomes
? Over 20 enzymes for activating the amino acids.
? Over 10 auxiliary proteins for the initiation,elongation
and termination of the polypeptide chains.
? Account for up to 90% of the chemical energy used by a
cell for all biosynthetic reactions.
? The molecules used for translation account for
more than 35% of the cell’s dry weight,
? However,proteins are synthesized with very high
efficiency,a complete polypeptide chain of 100
residues is synthesized in about 5 seconds in an
E.coli cells at 37oC.
2,The molecular mechanism of protein
synthesis was mainly revealed during the
2nd half of the 20th century
? Ribonucleoprotein particles (were later called ribosomes)
were revealed to be the site of protein synthesis in rat liver
cells,using radioactively labeled amino acids and
immediate subcellular fractionations (early 1950s,by
Zamecnik).
? Amino acids were found to be activated by attaching to a
special form of heat-stable RNA molecules (later called
tRNAs) before being incorporated into polypeptides (1950s,
by Hoagland and Zamecnik).
? Each tRNA molecule was found to function as an
adapter (originally hypothesized by Francis Crick),
carrying a specific amino acid with one site and
recognizing a specific site on a template with another site,
? The concept of messenger RNA (mRNA) was boldly
formulated by Jacob and Monod in 1961,a short-lived
RNA should serve as the information carrier between
gene and protein (to explain the quick induction of
proteins in E.coli).
? This bold hypothesis was quickly confirmed by studies
of E.coli cells infected by T2 phages,
Ribosomes were revealed to be the site of
protein synthesis in early 1950s (pulse
labeling with radioactive amino acids and
subcellular fractionations).
Crick’s
adapter
hypothesis
Hydrogen
bonds
3,Amino acids in a polypeptide chain
were found to be coded by groups of
three nucleotides in a mRNA
? Simple calculation indicated that three or more bases are
probably needed to specify one amino acid.
? Genetic studies of insertion,deletion,and substitution
mutants showed codons for amino acids are triplet of
nucleotides; codons do not overlap and there is no
punctuation between codons for successive amino acid
residues.
? The amino acid sequence of a polypeptide is defined by a
linear sequence of contiguous codons,the first codon
establishs a reading frame.
Genetic studies showed that genetic
codons are successive triplets of nucleotides
Altered amino acid sequences
Amino acid sequence studies of tobacco mosaic
virus mutants and abnormal hemoglobins
showed that alterations usually affected only
one single amino acid,genetic codes are
nonoverlapping.
Each mRNA molecule would have three
potential reading frames (but only one
usually codes for a polypeptide chain),
4,The genetic codes were deciphered by
simply using the in vitro protein synthesis
system
? Artificially synthesized poly(U),synthesized using
polynucleotide phosphorylase,was added to 20 reaction
tubes each containing the cell-free E.coli extract,GTP,
ATP,a mixture of 20 amino acids,and one 14C labeled
amino acid.
? Radioactive polypeptide was only detected in the tube
containing [14C]-Phe (with high concentration of Mg2+ ).
? When poly (A) and poly (C) were added,radioactive
polypeptides were only detected in the tubes containing
14C-labeled L-Lys and L-Pro respectively.
? UUU,AAA,CCC encodes Phe,Lys,Pro
respectively.
? When poly(G) was added,no polypeptides
synthesized prabably due to the formation of
tetraplexes of the poly (G) strands.
5,Base composition of the triplets coding
for some amino acids were revealed
using mixed copolymers of RNA
? The composition of an RNA synthesized using
polyribonucleotide phosphorylase depends on the
proportion of each NDP present in the reaction mixture.
? Investigation of the identity and quantity of the amino acids
incorporated into the polypeptides in response to random
polymers of RNA made from various ratios of NDPs can
reveal the nucleotide composition (but not exact sequence)
of the triplets corresponding to certain amino acids.
Composition of triplets coding for certain amino acids were assigned
6,Many trinucleotides were found to
promote the binding of specific
aminoacyl-tRNA to ribosomes
? It was discovered in 1964 that a specific aminoacyl-
tRNA would bind to the isolated ribosomes when
the corresponding synthetic polynucleotide
messenger or only the trinucleotide is present.
? Many genetic codes were revealed by examining
which aminoacyl-tRNA is bound to the ribosomes
mixed with specific trinucleotides using filter-
binding assay.
The filter-binding assay
for detecting the binding
of a trinucleotide to a
specific aminoacyl-tRNA
molecule,about 50
codons were assigned by
this simple and elegant
method.
7,Polyribonucleotides of defined
repeating sequences of two to four bases
helped to end the decoding work
? Khorana successfully developed a method to synthesize
polyribonucleotides of defined repeating sequences
using a combination of organic synthesis and enzymatic
techniques.
? Polypeptides synthesized using these
polyribonucleotides had repeating one to a few amino
acids.
? Sequences for specific genetic codes can be determined
by comparing the information obtained here and those
obtained by using RNAs having random sequences
made from two nucleotides of determined ratio.
Copolymer of repeating dinucleotides always
lead to synthesis of polypeptides of repeating
dipeptides:
ABABABABABABAB--
aa1---aa2---aa1---aa2----
Copolymer of repeating trinucleotides will lead
to the synthesis of three homopolypeptides:
ABCDABCDABCDABCDABCDABCDABCDABCD----
ABCDABCDABCDABCDABCDABCDABCDABCD----
ABCDABCDABCDABCDABCDABCDABCDABCD----
ABCDABCDABCDABCDABCDABCDABCDABCD----
Copolymer of repeating tetranucleotides will lead
to the synthesis of a single type of polypeptide with repeating
tetrapeptides.
Three different
homopolypeptides are
produced from most
polyribonucleotides
consisting of repeating
sequences of three
nucleotides;
one type of polypeptide
containing repeating
tetrapeptides was always
produced from
polyribonucleotides
consisting of repeating
sequences of four
nucleotides.
8,All 64 triplet codes were deciphered by
1966
? 61 of the codons code for the 20 amino acids and three
(UAA,UAG,UGA) for chain termination,called
termination codons,stop codons,or nonsense codons).
? AUG is a dual codon coding for initiation and Met.
? 18 of the amino acids are coded by more than one codon,
the genetic codes are degenerate.
? The codes seem to have evolved in such a way to minimize
the deleterious effects of mutations,especially at the third
bases,XYU and XYC always encode the same amino acid
XYA and XYG usually code for the same amino acid.
? A reading frame codes for more than 50
amino acids without a stop codon is called
an open reading frame,which has the
potential of encoding a protein.
All 64
genetic
codes
Established
the chemical
structure of
tRNA
Established the in
vitro system for
revealing the
genetic codes
Devised methods
to synthesize RNAs
with defined
sequences
9,The genetic code has been proved to
be nearly(not absolutely) universal
? Direct comparisons of the amino acid sequences of proteins
with the corresponding base sequence of their genes or
mRNAs,as well as recombinant DNA technologies,proved
that the genetic codes deciphered from in vitro studies were
correct and almost universally applicable.
? A small number of,unusual codes” have been revealed in
many mitochondria genomes and nuclear genome of a few
organisms,
10,Overlapping genes were found in
some viral DNAs
? Genes usually do not overlap.
? The 5.3 kb DNA of bacteriophage fX174 was found to be
not long enough to code for the ten proteins it produces.
? Detailed sequence correlation of the viral DNA and the
protein sequences revealed the,genes within genes”
phenomena.
? The overlapping genes use different reading frames.
? This phenomena was also found in other viruses (including
l phage,SV40).
Some genes
overlap in the
fX174
bacteriophage
DNA
11,Three kinds of RNA molecules
perform different but cooperative
functions in protein synthesis
? mRNAs carry the genetic information copied from DNA in
the form of genetic codons.
? tRNAs mediate the incorporation of specific amino acids
according to genetic codons present on the mRNA
molecules via their specific anticodon triplets.
? rRNAs associate with a set of proteins to form the protein-
synthesizing machines (ribosomes) and probably catalyze
peptide bond formation during protein synthesis.
Roles of the
3 types of
RNAs in
translation
All tRNAs have common structural features:
cloverleaf in secondary,“L” in 3-D structures.
12,Some tRNA molecules can recognize
more than one codons via wobble pairing
? The adapter tRNAs recognize the codons on a mRNA via a
triplet called anticodons.
? It was first proposed that a specific tRNA anticodon would
exist for every of the 61 (or 64) codons,but less tRNAs
were revealed.
? It was revealed that highly purified tRNA molecules (e.g.,
alanyl-tRNAAla) of known sequence could recognize several
different codons.
? Inosine,which may form base pair with A,U,and C,was
found to be present at the first position of the anticodons in
some tRNAs.
? Crick proposed the,wobble hypothesis” in 1966 to explain
the pairing features between anticodons and codons:
– The first two bases of a codon in mRNA confer most of
the coding specificity,the third base can be loosely
paired with the anticodons;
– The first base of some anticodons can wobble and
determines the number of codons a given tRNA can read
(A and C for one,U and G for two,I for three);
– Codons that specify the same amino acid but differ in
either of the first two bases need different tRNAs,i.e.,a
mininum of 31 tRNA are needed to translate the 61
codons;
? This hypothesis has been widely supported by all the
evidence gathered since (thus the,wobble rule”).
? This moderate pairing strength may serve to
optimize both the accuracy and speed of polypeptide
synthesis.
The codon-anticodon pairing between the a mRNA and a
tRNA,the presence of an inosinate residue at position one
in the anticodon allows the tRNA to recognize a few
codons.
I
I I
C
U
A
G U
Possible wobble pairing between anticodon and codon.
13,Ribosomes are the protein-
synthesizing machines
? All ribosomes consist of two units of unequal size.
? The large unit contain two or three rRNA molecules and 31
or 50 proteins.
? The small unit contain one rRNA molecule and 21 or 33
proteins.
? The total size of the prokaryotic and eukaryotic ribosomes
are 70S and 80S respectively.
? The rRNA and protein components of the bacterial
ribosomes have been separated and successfully
reconstituted in vitro.
Three rRNA
52 proteins
Four rRNA
83 proteins
Ribosomes are ribonucleoprotein particles for
synthesizing proteins.
Structure of
70S ribosome
at 5.5 A
50S
30S
14,Pulse-labeling (isotope tracer) studies
revealed that polypeptide synthesis
begins at the N-terminal
? 3H-leucine was added to reticulocyte cells actively
synthesizing hemoglobin for a short period of time.
? a and b chains of hemoglobin were isolated,treated with
trypsin and analyzed by fingerprinting and autoradiography.
? A gradient of radioactivity increasing from the amino to
carboxyl end of each chain was detected,indicating that the
carboxyl end was synthesized last,the polypeptide chain
grows by successive addition of amino acids at the C-
terminal.
Pulse-labeling (isotope tracer) studies revealed that
polypeptide synthesis begins at the N-terminal
15,mRNA is efficiently translated by
polysomes in the 5` 3` direction
? When the synthetic polynucleotide AAA(AAA)nAAC
was used as templates to guide polypeptide synthesis in
a cell-free protein-synthesizing system,the polypeptide
Lys-(Lys)n-Asn was produced.
? Translation undergoes from 5` to 3` along the mRNA.
? EM studies showed that multiple ribosomes (as
polysomes) can translate one single mRNA
simultaneously in all cells.
? Transcription and translation are closely coupled in
bacteria.
? Many eukaryotic polysomes are circular,which may
allow rapid recycling of ribosomes for translation.
Electron microscopic
examination of coupled
transcription and
translation in E.coli.
Direction of
transcription
A single mRNA is usually
translated by multiple
ribosomes (polysomes)
simultaneously.
Formation of circular eukaryotic mRNA by protein-
protein interactions of eIF4E and eIF4G (binding to
the m7G cap),poly(A)-binding protein I (PABI)
Force-field
electron micrograph
Model of protein synthesis on circular polysomes and
recycling of ribosomal subunits.
16,The synthesis of a protein can be
divided into five stages
? Each amino acid is first covalently attached to a specific
tRNA molecule in a reaction catalyzed by a specific
aminoacyl-tRNA synthetase (Stage 1).
? The mRNA then binds to the smaller subunit of the
ribosome,after which the initiating aminoacyl-tRNA and
the large subunits of the ribosome will bind in turn to form
the initiating complex (Stage 2)
? The first peptide bond is then formed after the second
aminoacyl-tRNA is recruited with help of the elongation
factors,and the chain is then further elongated (stage 3).
? When a stop codon (UAA,UAG,and UGA) is met,
the extension of the polypeptide chain will come to a
stop and is released from the ribosome with help
from release factors (Stage 4).
? The newly synthesized polypeptide chain has to be
folded and modified (in many cases) before
becoming a functional protein (Stage 5).
Each amino acid is
specifically attached
to a specific tRNA
before used for
protein synthesis
Stage 1
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.
Stage 2
AA2
Polypeptide chain
is elongated on
the ribosome
Stage 3
The polypeptide chain is
released from the ribosome
when meeting a stop
codon (UAA,UGA,or
UGA)
Stage 4
17,The 20 aminoacyl-tRNA synthetases
attach the 20 amino acids to one or more
specific tRNAs
? An amino acid is first activated to form an aminoacyl-AMP
intermediate (can be isolated when tRNA is absent),and is
then charged to one or more specific tRNAs all catalyzed by
one such specific aminoacyl-tRNA synthetase.
? The 20 synthetases have diverse sizes,subunit composition,
and amino acid sequences and are categorized into two
classes,class I and II,which bind to opposite faces of the
incoming tRNAs,link the amino acids to the 2`-OH and 3`-
OH groups of the terminal adenosine respectively.
Aminoacyl-tRNA synthetases
can be divided into two classes
based on differences in structure
and reaction mechanisms.
Aminoacyl-tRNA
synthetase
3 binding sites
in the active site
Aminoacyl-
AMP
Aminoacyl-
tRNA
Each synthetase
charges a
specific
tRNA with
a specific
amino acid
“the second
genetic codes”.
Gln-tRNA synthetase
(type I,monomeric)
Asp-tRNA synthetase
(type II,dimeric)
ATP
Amino acid arm
Anticodon arm
