? H1 The genetic code
? H2 Protein synthesis(translation) in
prokaryotes
? H3 Translation in eukaryotes
? H4 Protein targeting
? H5 Protein glycosylation
H protein synthesis
? Overview
? Restriction enzyme digestion
? Nomenclature
? Gel electrophoresis
? Restriction maps
? Restriction fragment length
polymorphisms(RFLP)
Restriction enzymes
? The genetic code is a triplet code
? The genetic code is degenerate
? Universality of the genetic code
? Reading frames
? Open reading frames
H1 The genetic code
Triplet code
? The nucleotide sequence of the mRNA
is colinear with the amino acid
sequence of the polypeptide in
encodes,The relationship between
them is called the genetic code
? 5’---3’ N terminal-----C terminal
Triplet code
? The three nucleotides in group
called codons
? 43=64 codons
? Initiation codon (AUG)
? stop codon ( UAA,UAG,UGA)
Triplet code
The genetic code is degenerate
? 64 codons,but only 20 amino acids,
so that,a single amino acid is coded
for by several different codons,that
is,degenerate,
? Synonyms:different codons specify
the same amino acid,
Triplet code
? Codon and anticodon,a triplet of bases
in a specific tRNA molecule,each base
is the codon base –pairs with its
complementary base in the anticodon,
? Wobble base-pairing
Degeneracy of the genetic code
Wobble rules
Universality of the genetic code
? All living organism used the
same code,
? But there are a few differences,
in mitochondrial mRNAs,some
codons have different
meanings,
Different code
Reading frames
Open reading frames
? An open reading frame (ORE) is a run
of codons that starts with ATG and
ends with a termination codon,TGA
TAA or TAG,Coding regions of genes
contain relatively long OPFs unlike
noncoding DNA where ORFs are
comparatively short,
? Overview
? Synthesis of aminoacyl-tRNA
? Initiation of protein synthesis
? Elongaion
? Termination
H2 translation in prokaryotes
Overview
? Translation relies upon
aminoacyl-tRNA that carry
specific amino acids and
recognize the corresponding
codons in mRNA by anticodon-
codon base-pairing,
Synthesis of aminoacyl-tRNA
(amino acid activation)
Each tRNA molecule has a cloverleaf
secondary structure consisting of three
stem loops,one of which bears the
anticodon at its end,The reaction is
called amino acid activation,
Synthesis of aminoacyl-tRNA is crucially
important for two reasons
? First, each amino acid must be covalently
linked to a tRNA molecule in order to take part
in protein synthesis,which depend upon the
adaptor function of tRNA,
? Second,the covalent bond is a high energy
bond that enables the amino acid to react with
the end of the growing polypeptide chain,
The synthesis reaction
occurs in two steps
? The first step is the reaction of amino acid
and ATP to form an aminoacyl-AMP,
? The second step is the aminoacyl group of
aminoacyl-AMP is transferred to the 3’end
of the tRNA molecule to form aminoacyl-
tRNA,
Step one
Step two,
Aminoacyl-AMP+tRNA-----aminoacyl-tRNA +AMP
Initiation of protein synthesis
fMet-tRNAfMet
Elongaion
Protein synthesis in E.coli
Initiation
? Each ribosome has three binding sites
for tRNAs; an A site where the incoming
aminoacyl-tRNA binds,a P site where the
tRNA linked to the growing polypeptide
chain is bound,and an E site which
binds tRNA prior to its release from the
ribosome,
? Translation in prokaryotes begins by the
formation of a 30s initiation complex
between the 30s ribosomal subunit,mRNA,
initiation factors and fMet tRNA fmet, The
30s subunit binds to the Shine-Dalgarno
sequence which lies 5' to the AUG Start
codon and is complementary to the 16s
rRNA of the small ribosomal subunit,
Initiation
? The ribosome then moves in a 3' direction
along the mRNA until it encounters the AUG
codon,The 50s ribosomal subunit now binds
to the 30s initiation complex to form the 70s
initiation complex,In this complex,the antico-
don of form the 70s initiation complex,In this
complex,the anticodon of the fMet tRNA fMet
is base-paired to the AUG initiation codon
(start codon) in the P site,
Initiation
Initiation
Elongation
? Elongation of the polypeptide
chain occurs in three steps,
? 1.Binding
? 2.Peptide bond formation
? 3.Translocation
Elongation
Elongation Factor Tu(EF-Tu)
Termination
? Initiation
? Elongation
? Termination
H3 Translation in eukaryotes
? Overview
? Secretory protein
? Plasma membrane proteins
? Proteins of the endoplasmic reticulum
? Lysosomal proteins
? Mitochondrial and chloroplast proteins
? Nuclear proteins
H4 Protein targeting
Overview
? Both in prokaryotes and eukaryotes,
newly synthesized proteins must be
delivered to specific subcellular
location or exported from the cell for
correct for activity,This
phenomenon is called protein
targeting,
Secretory proteins
? Secretory proteins have an N-
terminal signal peptide which targets
the protein to be synthesized on the
rough endoplasmic reticulum,
? During synthesis it is translocated
through the RER membrane into the
lumen,
? Vesicles then bud off from the RER and carry
the protein to the Golgi complex,where it
becomes glycosylated,
? Others vesicles then carry it to the plasma
membrane,
? Fusion of these transport vesicles with the
plasma membrane then releases the protein
to the cell exterior,
Secretory proteins
Plasma membrane proteins
? The orientation of the protein in the membrane
is determined by topogenic sequences within
the polypeptide chain,Type Ⅰ proteins have a
cleaved N-terminal signal sequence and a
hydrophobic stop-transfer sequence, Type Ⅱ
have an uncleaved N-terminal signal sequence
that doubles as the membrane-anchoring
sequence,and Type Ⅲ have multiple signal
sequences and stop-transfer sequences,
Proteins of the endoplasmic reticulum
? Proteins destined for the ER have an N-terminal
signal peptide,are synthesized on the RER,are
translocated into the RER lumen and transport-
ed by vesicles to the Golgi,Once there,a C-
terminal amino acid sequence （ KDEL ） is
recognized by a Golgi receptor protein that
causes other vesicles to return the protein to
the ER,
Lysosomal proteins
? Lysosomal proteins are targeted to
the lysosomes via the addition of a
mannose 6-phosphate signal that is
addad in the cis-compartment of the
Golgi and is recognized by a receptor
protein in the trans-compartment of
the Golgi,
Mitochondrial and chloroplast
proteins
? Most mitochondria and chloroplast protein
are made on free cytosolic ribosomes,
released into the mitochondrial matrix
requires a matrix-targeting sequence and
occurs at sites where the outer and inner
mitochondrial membranes come into
contact,
? Three types of protein
glycosylation
? Synthesis of O-linked
oligosaccharides
? Synthesis of N-linked
oligosaccharides
H5 protein glycosylation
Three types of protein
glycosylation
? Many protein synthesized by ribosomes of
the RER contain short chains of
carbohydrates (oligosaccharides )and are
called glycoproteins,The oligosaccharides
are of two main types; O-linked (to the
Oxyside chain of Ser or Thr) and N-linked
(to the NH2 side chain of Asn),
Synthesis of O-linked
oligosaccharides
? O-linked oligosaccharides are
Synthesized by the sequential
addition of monosaccharides to
the protein as it passes through
the Golgi complex,
Synthesis of N-linked
oligosaccharides
? All N-linked oligosaccharides have a
common pentasaccharide core structure
of three mannose residues and two N-
acetylglucosamine(GlcNAc) residues,The
oligosaccharide is initially synthesized on
a dolichol phosphate carrier that is
anchored to the RER membrane,
This is then transferred to the protein
and subsequently trimmed during
passage of the protein through the
RER and Golgi complex,Additional
monosaccharides are added in the
Golgi to produce either a high
mannose type oligosaccharide or a
complex type oligosaccharide,
? H2 Protein synthesis(translation) in
prokaryotes
? H3 Translation in eukaryotes
? H4 Protein targeting
? H5 Protein glycosylation
H protein synthesis
? Overview
? Restriction enzyme digestion
? Nomenclature
? Gel electrophoresis
? Restriction maps
? Restriction fragment length
polymorphisms(RFLP)
Restriction enzymes
? The genetic code is a triplet code
? The genetic code is degenerate
? Universality of the genetic code
? Reading frames
? Open reading frames
H1 The genetic code
Triplet code
? The nucleotide sequence of the mRNA
is colinear with the amino acid
sequence of the polypeptide in
encodes,The relationship between
them is called the genetic code
? 5’---3’ N terminal-----C terminal
Triplet code
? The three nucleotides in group
called codons
? 43=64 codons
? Initiation codon (AUG)
? stop codon ( UAA,UAG,UGA)
Triplet code
The genetic code is degenerate
? 64 codons,but only 20 amino acids,
so that,a single amino acid is coded
for by several different codons,that
is,degenerate,
? Synonyms:different codons specify
the same amino acid,
Triplet code
? Codon and anticodon,a triplet of bases
in a specific tRNA molecule,each base
is the codon base –pairs with its
complementary base in the anticodon,
? Wobble base-pairing
Degeneracy of the genetic code
Wobble rules
Universality of the genetic code
? All living organism used the
same code,
? But there are a few differences,
in mitochondrial mRNAs,some
codons have different
meanings,
Different code
Reading frames
Open reading frames
? An open reading frame (ORE) is a run
of codons that starts with ATG and
ends with a termination codon,TGA
TAA or TAG,Coding regions of genes
contain relatively long OPFs unlike
noncoding DNA where ORFs are
comparatively short,
? Overview
? Synthesis of aminoacyl-tRNA
? Initiation of protein synthesis
? Elongaion
? Termination
H2 translation in prokaryotes
Overview
? Translation relies upon
aminoacyl-tRNA that carry
specific amino acids and
recognize the corresponding
codons in mRNA by anticodon-
codon base-pairing,
Synthesis of aminoacyl-tRNA
(amino acid activation)
Each tRNA molecule has a cloverleaf
secondary structure consisting of three
stem loops,one of which bears the
anticodon at its end,The reaction is
called amino acid activation,
Synthesis of aminoacyl-tRNA is crucially
important for two reasons
? First, each amino acid must be covalently
linked to a tRNA molecule in order to take part
in protein synthesis,which depend upon the
adaptor function of tRNA,
? Second,the covalent bond is a high energy
bond that enables the amino acid to react with
the end of the growing polypeptide chain,
The synthesis reaction
occurs in two steps
? The first step is the reaction of amino acid
and ATP to form an aminoacyl-AMP,
? The second step is the aminoacyl group of
aminoacyl-AMP is transferred to the 3’end
of the tRNA molecule to form aminoacyl-
tRNA,
Step one
Step two,
Aminoacyl-AMP+tRNA-----aminoacyl-tRNA +AMP
Initiation of protein synthesis
fMet-tRNAfMet
Elongaion
Protein synthesis in E.coli
Initiation
? Each ribosome has three binding sites
for tRNAs; an A site where the incoming
aminoacyl-tRNA binds,a P site where the
tRNA linked to the growing polypeptide
chain is bound,and an E site which
binds tRNA prior to its release from the
ribosome,
? Translation in prokaryotes begins by the
formation of a 30s initiation complex
between the 30s ribosomal subunit,mRNA,
initiation factors and fMet tRNA fmet, The
30s subunit binds to the Shine-Dalgarno
sequence which lies 5' to the AUG Start
codon and is complementary to the 16s
rRNA of the small ribosomal subunit,
Initiation
? The ribosome then moves in a 3' direction
along the mRNA until it encounters the AUG
codon,The 50s ribosomal subunit now binds
to the 30s initiation complex to form the 70s
initiation complex,In this complex,the antico-
don of form the 70s initiation complex,In this
complex,the anticodon of the fMet tRNA fMet
is base-paired to the AUG initiation codon
(start codon) in the P site,
Initiation
Initiation
Elongation
? Elongation of the polypeptide
chain occurs in three steps,
? 1.Binding
? 2.Peptide bond formation
? 3.Translocation
Elongation
Elongation Factor Tu(EF-Tu)
Termination
? Initiation
? Elongation
? Termination
H3 Translation in eukaryotes
? Overview
? Secretory protein
? Plasma membrane proteins
? Proteins of the endoplasmic reticulum
? Lysosomal proteins
? Mitochondrial and chloroplast proteins
? Nuclear proteins
H4 Protein targeting
Overview
? Both in prokaryotes and eukaryotes,
newly synthesized proteins must be
delivered to specific subcellular
location or exported from the cell for
correct for activity,This
phenomenon is called protein
targeting,
Secretory proteins
? Secretory proteins have an N-
terminal signal peptide which targets
the protein to be synthesized on the
rough endoplasmic reticulum,
? During synthesis it is translocated
through the RER membrane into the
lumen,
? Vesicles then bud off from the RER and carry
the protein to the Golgi complex,where it
becomes glycosylated,
? Others vesicles then carry it to the plasma
membrane,
? Fusion of these transport vesicles with the
plasma membrane then releases the protein
to the cell exterior,
Secretory proteins
Plasma membrane proteins
? The orientation of the protein in the membrane
is determined by topogenic sequences within
the polypeptide chain,Type Ⅰ proteins have a
cleaved N-terminal signal sequence and a
hydrophobic stop-transfer sequence, Type Ⅱ
have an uncleaved N-terminal signal sequence
that doubles as the membrane-anchoring
sequence,and Type Ⅲ have multiple signal
sequences and stop-transfer sequences,
Proteins of the endoplasmic reticulum
? Proteins destined for the ER have an N-terminal
signal peptide,are synthesized on the RER,are
translocated into the RER lumen and transport-
ed by vesicles to the Golgi,Once there,a C-
terminal amino acid sequence （ KDEL ） is
recognized by a Golgi receptor protein that
causes other vesicles to return the protein to
the ER,
Lysosomal proteins
? Lysosomal proteins are targeted to
the lysosomes via the addition of a
mannose 6-phosphate signal that is
addad in the cis-compartment of the
Golgi and is recognized by a receptor
protein in the trans-compartment of
the Golgi,
Mitochondrial and chloroplast
proteins
? Most mitochondria and chloroplast protein
are made on free cytosolic ribosomes,
released into the mitochondrial matrix
requires a matrix-targeting sequence and
occurs at sites where the outer and inner
mitochondrial membranes come into
contact,
? Three types of protein
glycosylation
? Synthesis of O-linked
oligosaccharides
? Synthesis of N-linked
oligosaccharides
H5 protein glycosylation
Three types of protein
glycosylation
? Many protein synthesized by ribosomes of
the RER contain short chains of
carbohydrates (oligosaccharides )and are
called glycoproteins,The oligosaccharides
are of two main types; O-linked (to the
Oxyside chain of Ser or Thr) and N-linked
(to the NH2 side chain of Asn),
Synthesis of O-linked
oligosaccharides
? O-linked oligosaccharides are
Synthesized by the sequential
addition of monosaccharides to
the protein as it passes through
the Golgi complex,
Synthesis of N-linked
oligosaccharides
? All N-linked oligosaccharides have a
common pentasaccharide core structure
of three mannose residues and two N-
acetylglucosamine(GlcNAc) residues,The
oligosaccharide is initially synthesized on
a dolichol phosphate carrier that is
anchored to the RER membrane,
This is then transferred to the protein
and subsequently trimmed during
passage of the protein through the
RER and Golgi complex,Additional
monosaccharides are added in the
Golgi to produce either a high
mannose type oligosaccharide or a
complex type oligosaccharide,
