2010-5-14
1
Welcome Each of You
to My Molecular
Biology Class
2010-5-14
2
Molecular Biology of the Gene,
5/E --- Watson et al,(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the Genome
Part III,Expression of the Genome
Part IV,Regulation
Part V,Methods
2010-5-14
3
Part III,Expression of the
Genome
?This part concerned with one
of the greatest challenges in
understanding the gene－
how the gene is expressed.
2010-5-14
4
Part III,Expression of the Genome
Ch 12,Mechanisms of
transcription
Ch 13,RNA splicing
Ch 14,Translation
Ch 15,The genetic code
DNA RNA protein
translation
2010-5-14 5
Chapter 14,
Translation
2010-5-14
6
Translation extremely
costs
In rapid growing bacterial cells,
protein synthesis consumes
?80% of the cell’s energy
?50% of the cell’s cell’s dry
weight
Why?
2010-5-14
7
The main challenge of
translation
?The genetic information in
mRNA cannot be recognized by
amino acids,
?The genetic code has to be
recognized by an adaptor
molecular (translator),and this
adaptor has to accurately recruit
the corresponding amino acid,
2010-5-14
8
Translation machinery
1,mRNAs (~5% of total cellular
RNA)
2,tRNAs (~15%)
3,aminoacyl-tRNA synthetases
(氨酰 tRNA合成酶 )
4,ribosomes (~100 proteins and
3-4 rRNAs--~80%)
2010-5-14
9
Outline
Topics 1-4,Four components of
translation machinery,
T1-mRNA; T2-tRNA; T3-Attachment
of amino acids to tRNA (aminoacyl-
tRNA synthetases); T4-The
ribosome
Topic 5-6,Translation process.
T5-initiation; T6-elongation; T7-
termination,
Topic 8,Translation-dependent
regulation of mRNA and protein
stability
2010-5-14
10
Topic 1,mRNA
?Only a portion of each mRNA
can be translated.
?The protein-coding region of the
mRNA consists of an ordered
series of 3-nt-long units called
codons that specify the order of
amino acids.
2010-5-14
11
1-1 polypeptide chains
are specified by ORF
? The protein coding region of each
mRNA is composed of a contiguous,
non-overlapping string of codons
called an opening reading frame
(ORF),
? An ORF should begins with a start
codon and end with stop codon.
? mRNA containing more than one ORF
is called polycistronic mRNAs.
Mes
sa
ge
R
NA
2010-5-14
12
Fig 14-1 Three possible reading
frames of the E,coli trp leader
sequence
2010-5-14
13
1-2 Prokaryotic mRNAs
have a ribosome binding
site that recruits the
translational machinery
Mes
sa
ge
RNA
1-3 Eukaryotic mRNA
are modified at their 5’
and 3’ ends to facilitate
translation,
2010-5-14
14
? Ribosome binding site (RBS) or SD-
sequence in prokaryotic mRNA,
complementary with the sequence at the
3’ end of 16S rRNA,
Fig 14-2-a structure of mRNA
2010-5-14
15
Once
Kozak sequence
? Eukaryotic mRNA uses a methylated
cap to recruit the ribosome,Once bound,
the ribosome scans the mRNA in a 5’-3’
direction to find the AUG start codon.
? Kozak sequence increases the
translation efficiency.
? Poly-A in the 3’ end promotes the
efficient recycling of ribosomes
Fig 14-2-b
2010-5-14
16
Topic 2,tRNA
At the heart of protein synthesis
is the translation of nucleotide
sequence information into amino
acids,This work is accomplished
by tRNA.
2010-5-14
17
1,The are many types of tRNA
molecules in cell (~40).
2,Each tRNA molecule is attached to a
specific amino acids (20) and each
recognizes a particular codon,or
codons (61),in the mRNA
3,All tRNAs end with the sequence 5’-
CCA-3’ at the 3’ end,where the
aminoacyl tRNA synthetase adds the
amino acid.
2-1,tRNA are adaptors between
codons and amino acids
TR
ANS
FE
R
RNA
2010-5-14
18
1,tRNAs are 75-95 nt in length,
2,There are 15 invariant and 8 semi-invariant
residues,The position of invariant and
semi-variant nucleosides play a role in
either the secondary and tertiary structure,
3,There are many modified bases,which
sometimes accounting for 20% of the total
bases in one tRNA molecule,Over 50
different types of them have been
observed.
Primary structure
2010-5-14
19
Fig 14-3 unusual bases
4,Pseudouridine (?U) is a modified base,
These modified bases in tRNA lead to
improved tRNA function
2010-5-14
20
? The cloverleaf structure is a
common secondary structural
representation of tRNA
molecules which shows the base
paring of various regions to
form four stems (arms) and
three loops,
2-2,tRNAs share a common
secondary structure that
resemble a cloverleaf
TR
ANS
FE
R
RNA
2010-5-14
21
Fig 14-4 the secondary structure
2010-5-14
22
tRNA
secondary
structure
D loop T loop
Anticodon loop
2010-5-14
23
? The 5’-and
3’-end are
largely base-
paired to
form the
amino acid
acceptor
stem which
has no loop,
Amino acid acceptor stem:
2010-5-14
24
? Composed
of 3 or 4 bp
stem and a
loop called the
D-loop (DHU-
loop) usually
containing the
modified base
dihydrouracil.
D-arm and D-loop
2010-5-14
25
Consisting of a 5
bp stem and a 7
residues loop in
which the
anticodon is
located,The 3-nt
anticodon
sequence is used
to recognize the
codon sequence
in the mRNA
Anticodon loop and anticodon loop
2010-5-14
26
Variable arm and T-arm:
Variable arm,3 to
21 residues and may
form a stem of up to
7 bp.
T-arm is composed
of a 5 bp stem
ending in a loop
containing the
invariant residues
GT?C,
2010-5-14
27
2-3,tRNAs have an L-
shaped 3-D structure
TR
ANS
FE
R
RNA
Fig 14-5 the 3-D structure of tRNA
2010-5-14
28
Formation:
9 hydrogen bones (tertiary
hydrogen bones) help the
formation of tRNA tertiary
structure,mainly involving in
the base paring between the
invariant bases,
2010-5-14
29
?Base pairing between residues in
the D-and T-arms fold the tRNA
molecule into an L-shape,with
the anticodon loop at one end
and the amino acid acceptor site
at the other (Fig,14-5),The
base pairing is strengthened by
base stacking interactions,
2010-5-14
30
Topic 3,attachment of
amino acids to tRNA
?Amino acids should attach to
tRNA first before adding to
polypeptide chain,
?tRNA molecules to which an
amino acid is attached are said
to be charged,and tRNAs
lacking an amino acid are said
to uncharged,
2010-5-14
31
3-1 tRNAs are charged by
attachment of an amino acid
to the 3’ terminal A of the
tRNA via a high energy acyl
linkage
?The energy released when the
high-energy bond is broken
helps drive the peptide bond
formation during protein
synthesis.
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
32
3-2 Aminoacyl tRNA
synthetases charge tRNA in
two steps
1,Adenylylation (腺苷酰化 ) of
amino acids,transfer of AMP to
the COO- end of the amino acids.
2,tRNA charging,transfer of the
adenylylated amino acids to the
3’ end of tRNA,generating
aminoacyl-tRNAs.
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
33
? Reaction step:
First,the aminoacyl-
tRNA synthetase
attaches AMP to
the-COOH group of
the amino acid
utilizing ATP to
create an
aminoacyl (氨酰的 )
adenylate (腺苷酸 )
intermediate.
Then,the
appropriate tRNA
displaces the AMP,
Also see Figure 14-6 in your text book
2010-5-14
34
Proofreading
? Proofreading occurs at step 2 when a
synthetase carries out step 1 of the
aminoacylation reaction with the
wrong,but chemically similar,amino
acid,
? Synthetase will not attach the
aminoacyl adenylate to the cognate
tRNA,but hydrolyze the aminoacyl
adenylate instead.
2010-5-14
35
3-3,each aminoacyl tRNA
synthetase attaches a single
amino acids to one or more
tRNAs---accurate charging is
essential
? Each of the 20 amino acids is
attached to the appropriate tRNA (s)
by aminoacyl-tRNA synthetases.
? Most amino acids are specified by
more than one codon,and by more
than one tRNA as well,
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
36
? The same synthetase is responsible for
charging all tRNAs for a particular
amino acids.
? Consequently,most organisms have
20 synthetases for 20 different amino
acids.
2010-5-14
37
? Nomenclature of tRNA-
synthetases and charged tRNAs
Amino acid,serine
Cognate tRNA,tRNASer
Cognate aminoacyl-tRNA synthetase,
Ser-tRNA synthetase
Aminoacyl-tRNA,Ser-tRNASer
tRNA charging by Aminoacyl-tRNA
synthetases is specific
2010-5-14
38
Class I,attach the amino acids to the
2’OH of the tRNA,and is usually
monomeric.
Class II,attach the amino acids to
the 3’OH of the tRNA,and is usually
dimeric or tetrameric
There are two classes of tRNA
synthetases.
2010-5-14
39
3-4 tRNA synthetases
recognize unique structure
features of cognate tRNAs
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
? The recognition has to ensure two
levels of accuracy,(1) each tRNA
synthetase must recognize the
correct set of tRNAs for a
particular amino acids; (2) each
synthetase must charge all of
these isoaccepting tRNAs (即由一种
synthetase所识别的不同 tRNAs)
2010-5-14
40
? The specificity determinants
for accurate recognition are
clusters at two distinct sites,
the acceptor stem and the anti-
codon loop.
2010-5-14
41
Fig 14-8
Fig 14-7
2010-5-14
42
3-5 Aminoacyl-tRNA
formation is very accurate,
selection of the correct
amino acid
? The aminoacyl tRNA synthetases
discriminate different amino acids
according to different natures of their
side-chain groups.
? Some enzymes have editing pocket
to do proofreading by matching the
wrong product and hydrolyzing it (3-
6).
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
43
Ribosome is responsible to
place the charged tRNAs onto
mRNA through base pairing of
the codon in mRNA and
anticodon in tRNA
3-7 Ribosomes is unable to
discriminate between
correctly or incorrectly
charged tRNAs (是否携带正确
的氨基酸 )
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
44
Topic 4,the ribosome
Fig 14-17 two views of the ribosome
2010-5-14
45
4-1 the ribosome is composed
of a large and a small subunit
? The large subunit contains the
peptidyl transferase center,which is
responsible for the formation of
peptide bonds.
? The small subunit interacting with
mRNA contains the decoding center,
in which charged tRNAs read or
“decode” the codon units of the
mRNA.
RI
BO
SO
ME
S
2010-5-14
46
Fig 14-13** Ribosome
2010-5-14
47
4-2,the large and the small
subunits undergone
association and dissociation
during each cycle of
translation
RI
BO
SO
ME
S
2010-5-14
48
?Ribosome cycles,In cells,the
small and large ribosome subunits
associate with each other and the
mRNA,translate it,and then
dissociate after each round of
translation,This sequence of
association and dissociation is
called the ribosome cycle.
2010-5-14
49
Fig 14-14 Overview of the events of
translation/ribosome cycle
2010-5-14
50
Polysome/polyribosome,an
mRNA bearing multiple ribosomes
? Each mRNA can be translated
simultaneously by multiple ribosomes
Fig 14-15 A polyribosome
2010-5-14
51
RI
BO
SO
ME
S
4-3 New amino acids are
attached to the C-terminus of
the growing polypeptide
chain.
Protein is synthesized in a N-
to C- terminal direction
4-4 Peptide bonds are formed
by transfer of the growing
peptide chain from peptidyl-
tRNA to aminoacyl-tRNA.
2010-5-14
52
Fig 14-16
2010-5-14
53
The structure of the ribosome
4-5 Ribosomal RNAs are both
structural and catalytic
determinants of the ribosomes
4-6 The ribosome has three
binding site for tRNA.
4-7 Channels through the
ribosome allow the mRNA and
growing polypeptide to enter
and/or exit the ribosome.
RI
BO
SO
ME
S
2010-5-14
54F
ig 1
4-
19
3
-D
st
ru
ct
ur
e
of
th
e
ribo
so
me
inc
ludi
ng
3 bo
un
d
tR
NA
2010-5-14
55
Three binding site for tRNAs
Fig 14-18 A site,to bind the
aminoacylated-tRNA
B-site,to bind the
peptidyl-tRNA
E-site,to bind the
uncharged tRNA
2010-5-14
56
Channels for mRNA entering and
exiting are located in the small
subunit (see Fig,14-18)
There is a
pronounced kink in
the mRNA between
the two codons at P
and A sites,This kink
places the vacant A
site codon for
aminoacyl-tRNA
interaction,
Fig 14-20
2010-5-14
57
Channel for polypeptide chain
exiting locates in the large
subunit (see Fig,14-18)
The size of the
channel only
allow a very
limited folding
of the newly
synthesized
polypeptide
Fig 14-21
2010-5-14
58
? We now know that the rRNAs are not
simply structural components of the
ribosomes,Rather,they account for the
key function of the ribosomes,
? Most ribosomal proteins are on the
periphery of the ribosomes,not in its
interior,
? So,it’s inferred that the contemporary
ribosome evolved from a primitive
protein synthesis machine that was
composed entirely of RNA.
2010-5-14
59
Translation process
T5,Initiation of translation
T6,Elongation of translation
T7,termination of translation
Watch the animation
on your study CD
2010-5-14
60
Questions
1,Compare the mechanism of
translation initiation in prokaryotes
and eukaryotes (similarity and
difference)
2,How do prokaryotes and eukaryotes
find the translation start sites?
3,How do aminoacyl-tRNA
synthetases and the ribosomes
contribute to the fidelity of
translation,respectively?
2010-5-14
61
Overview of the events of
translation
Termination Elongation
Initiation
Fig 14-14
2010-5-14
62
5-6 Translation initiation factors
hold eukaryotic mRNAs in circles
Try to explain how the mRNA poly-A tail
contributes to the translation efficiency?
INI
TI
AT
IO
N
OF
T
RAN
SL
AT
IO
N
Fig 14-29
2010-5-14
63
6-3 Ribosome is a ribozyme
EL
ONG
AT
IO
N
OF
T
RAN
SL
AT
IO
N
Fig 14-33
Catalysis requires distance in the 1-3 ?,
and only RNA residues are present 18 ?
from the active site.
2010-5-14
64
EF-G mimics a tRNA molecule so as to
displace the tRNA bound to the A site
EF-G-GDPEF-Tu-GDPNP-Phe-tRNA
Fig 14-35
2010-5-14
65
Topic 8,translation dependent
regulation of mRNA and
protein stability
?Here regulation refers
cellular processes that deal
with defective mRNA and
their translated product.
2010-5-14
66
8-1,The SsrA RNA rescues (拯救 )
ribosomes that translate broken
mRNAs lacking a stop codon
(prokaryotes)
1.The ribosomes are trapped or
stalled on the broken mRNA
lacking a stop codon
2.The stalled ribosomes are
rescued by the action of a
chimeric RNA molecule that is
part tRNA and part mRNA,called
tmRNA.
3.SsrA is a 457-nt tmRNA
2010-5-14
67
Fig 14-39 SsrA rescues the stalled ribosomes
2010-5-14
68
8-2,Eukaryotic cells degrade
mRNAs that are incomplete or
have premature stop codons
Translation is tightly linked to the
process of mRNA decay in
eukaryotic cells
2010-5-14
69
When an mRNA contains a premature
stop codon (nonsense codon),the
mRNA is rapidly degraded by
nonsense mediated mRNA decay.
Pre-releasing the ribosome at the
nonsense codon prior to reaching the
exon-junction complex initiates a talk
between the complex and ribosome
to remove the 5’ cap from the mRNA
Nonsense mediated mRNA decay
2010-5-14
70
1,Translation of a normal
eukaryotic mRNA displace all
the exon junction complex
Fig 14-40a
2010-5-14
71
2,Nonsense mediated mRNA
decay
Fig 14-40b
2010-5-14
72
Non-stop mediated mRNA decay
rescues ribosomes that translate
mRNAs lacking a stop codon
(1)The lack of a stop codon results in
ribosome translation into the poly-A tail
to produce poly-Lys at the C-terminus
of the polypeptide; the poly-Lys marks
the newly synthesized for rapid
degradation,
Nonstop mediated decay
2010-5-14
73Fig 14-40c
(2)The ribosome eventually stalls at the 3’ end of
the mRNA,which is bound by the Ski7 protein
that triggers the ribosome dissociation and
recruits a 3’-5’ exonuclease activity to degrade
the,nonstop” mRNA,
2010-5-14
74
1,The main challenge of translation and
the solution
2,The structure and function of four
components of the translation
machinery.
3,Translation initiation,elongation and
termination (具体过程和翻译因子的作用 )
4,The mRNA and protein stability
dependent on translation (生物学问题是
什么，怎么解决的 )
Key points of the chapter
1
Welcome Each of You
to My Molecular
Biology Class
2010-5-14
2
Molecular Biology of the Gene,
5/E --- Watson et al,(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the Genome
Part III,Expression of the Genome
Part IV,Regulation
Part V,Methods
2010-5-14
3
Part III,Expression of the
Genome
?This part concerned with one
of the greatest challenges in
understanding the gene－
how the gene is expressed.
2010-5-14
4
Part III,Expression of the Genome
Ch 12,Mechanisms of
transcription
Ch 13,RNA splicing
Ch 14,Translation
Ch 15,The genetic code
DNA RNA protein
translation
2010-5-14 5
Chapter 14,
Translation
2010-5-14
6
Translation extremely
costs
In rapid growing bacterial cells,
protein synthesis consumes
?80% of the cell’s energy
?50% of the cell’s cell’s dry
weight
Why?
2010-5-14
7
The main challenge of
translation
?The genetic information in
mRNA cannot be recognized by
amino acids,
?The genetic code has to be
recognized by an adaptor
molecular (translator),and this
adaptor has to accurately recruit
the corresponding amino acid,
2010-5-14
8
Translation machinery
1,mRNAs (~5% of total cellular
RNA)
2,tRNAs (~15%)
3,aminoacyl-tRNA synthetases
(氨酰 tRNA合成酶 )
4,ribosomes (~100 proteins and
3-4 rRNAs--~80%)
2010-5-14
9
Outline
Topics 1-4,Four components of
translation machinery,
T1-mRNA; T2-tRNA; T3-Attachment
of amino acids to tRNA (aminoacyl-
tRNA synthetases); T4-The
ribosome
Topic 5-6,Translation process.
T5-initiation; T6-elongation; T7-
termination,
Topic 8,Translation-dependent
regulation of mRNA and protein
stability
2010-5-14
10
Topic 1,mRNA
?Only a portion of each mRNA
can be translated.
?The protein-coding region of the
mRNA consists of an ordered
series of 3-nt-long units called
codons that specify the order of
amino acids.
2010-5-14
11
1-1 polypeptide chains
are specified by ORF
? The protein coding region of each
mRNA is composed of a contiguous,
non-overlapping string of codons
called an opening reading frame
(ORF),
? An ORF should begins with a start
codon and end with stop codon.
? mRNA containing more than one ORF
is called polycistronic mRNAs.
Mes
sa
ge
R
NA
2010-5-14
12
Fig 14-1 Three possible reading
frames of the E,coli trp leader
sequence
2010-5-14
13
1-2 Prokaryotic mRNAs
have a ribosome binding
site that recruits the
translational machinery
Mes
sa
ge
RNA
1-3 Eukaryotic mRNA
are modified at their 5’
and 3’ ends to facilitate
translation,
2010-5-14
14
? Ribosome binding site (RBS) or SD-
sequence in prokaryotic mRNA,
complementary with the sequence at the
3’ end of 16S rRNA,
Fig 14-2-a structure of mRNA
2010-5-14
15
Once
Kozak sequence
? Eukaryotic mRNA uses a methylated
cap to recruit the ribosome,Once bound,
the ribosome scans the mRNA in a 5’-3’
direction to find the AUG start codon.
? Kozak sequence increases the
translation efficiency.
? Poly-A in the 3’ end promotes the
efficient recycling of ribosomes
Fig 14-2-b
2010-5-14
16
Topic 2,tRNA
At the heart of protein synthesis
is the translation of nucleotide
sequence information into amino
acids,This work is accomplished
by tRNA.
2010-5-14
17
1,The are many types of tRNA
molecules in cell (~40).
2,Each tRNA molecule is attached to a
specific amino acids (20) and each
recognizes a particular codon,or
codons (61),in the mRNA
3,All tRNAs end with the sequence 5’-
CCA-3’ at the 3’ end,where the
aminoacyl tRNA synthetase adds the
amino acid.
2-1,tRNA are adaptors between
codons and amino acids
TR
ANS
FE
R
RNA
2010-5-14
18
1,tRNAs are 75-95 nt in length,
2,There are 15 invariant and 8 semi-invariant
residues,The position of invariant and
semi-variant nucleosides play a role in
either the secondary and tertiary structure,
3,There are many modified bases,which
sometimes accounting for 20% of the total
bases in one tRNA molecule,Over 50
different types of them have been
observed.
Primary structure
2010-5-14
19
Fig 14-3 unusual bases
4,Pseudouridine (?U) is a modified base,
These modified bases in tRNA lead to
improved tRNA function
2010-5-14
20
? The cloverleaf structure is a
common secondary structural
representation of tRNA
molecules which shows the base
paring of various regions to
form four stems (arms) and
three loops,
2-2,tRNAs share a common
secondary structure that
resemble a cloverleaf
TR
ANS
FE
R
RNA
2010-5-14
21
Fig 14-4 the secondary structure
2010-5-14
22
tRNA
secondary
structure
D loop T loop
Anticodon loop
2010-5-14
23
? The 5’-and
3’-end are
largely base-
paired to
form the
amino acid
acceptor
stem which
has no loop,
Amino acid acceptor stem:
2010-5-14
24
? Composed
of 3 or 4 bp
stem and a
loop called the
D-loop (DHU-
loop) usually
containing the
modified base
dihydrouracil.
D-arm and D-loop
2010-5-14
25
Consisting of a 5
bp stem and a 7
residues loop in
which the
anticodon is
located,The 3-nt
anticodon
sequence is used
to recognize the
codon sequence
in the mRNA
Anticodon loop and anticodon loop
2010-5-14
26
Variable arm and T-arm:
Variable arm,3 to
21 residues and may
form a stem of up to
7 bp.
T-arm is composed
of a 5 bp stem
ending in a loop
containing the
invariant residues
GT?C,
2010-5-14
27
2-3,tRNAs have an L-
shaped 3-D structure
TR
ANS
FE
R
RNA
Fig 14-5 the 3-D structure of tRNA
2010-5-14
28
Formation:
9 hydrogen bones (tertiary
hydrogen bones) help the
formation of tRNA tertiary
structure,mainly involving in
the base paring between the
invariant bases,
2010-5-14
29
?Base pairing between residues in
the D-and T-arms fold the tRNA
molecule into an L-shape,with
the anticodon loop at one end
and the amino acid acceptor site
at the other (Fig,14-5),The
base pairing is strengthened by
base stacking interactions,
2010-5-14
30
Topic 3,attachment of
amino acids to tRNA
?Amino acids should attach to
tRNA first before adding to
polypeptide chain,
?tRNA molecules to which an
amino acid is attached are said
to be charged,and tRNAs
lacking an amino acid are said
to uncharged,
2010-5-14
31
3-1 tRNAs are charged by
attachment of an amino acid
to the 3’ terminal A of the
tRNA via a high energy acyl
linkage
?The energy released when the
high-energy bond is broken
helps drive the peptide bond
formation during protein
synthesis.
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
32
3-2 Aminoacyl tRNA
synthetases charge tRNA in
two steps
1,Adenylylation (腺苷酰化 ) of
amino acids,transfer of AMP to
the COO- end of the amino acids.
2,tRNA charging,transfer of the
adenylylated amino acids to the
3’ end of tRNA,generating
aminoacyl-tRNAs.
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
33
? Reaction step:
First,the aminoacyl-
tRNA synthetase
attaches AMP to
the-COOH group of
the amino acid
utilizing ATP to
create an
aminoacyl (氨酰的 )
adenylate (腺苷酸 )
intermediate.
Then,the
appropriate tRNA
displaces the AMP,
Also see Figure 14-6 in your text book
2010-5-14
34
Proofreading
? Proofreading occurs at step 2 when a
synthetase carries out step 1 of the
aminoacylation reaction with the
wrong,but chemically similar,amino
acid,
? Synthetase will not attach the
aminoacyl adenylate to the cognate
tRNA,but hydrolyze the aminoacyl
adenylate instead.
2010-5-14
35
3-3,each aminoacyl tRNA
synthetase attaches a single
amino acids to one or more
tRNAs---accurate charging is
essential
? Each of the 20 amino acids is
attached to the appropriate tRNA (s)
by aminoacyl-tRNA synthetases.
? Most amino acids are specified by
more than one codon,and by more
than one tRNA as well,
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
36
? The same synthetase is responsible for
charging all tRNAs for a particular
amino acids.
? Consequently,most organisms have
20 synthetases for 20 different amino
acids.
2010-5-14
37
? Nomenclature of tRNA-
synthetases and charged tRNAs
Amino acid,serine
Cognate tRNA,tRNASer
Cognate aminoacyl-tRNA synthetase,
Ser-tRNA synthetase
Aminoacyl-tRNA,Ser-tRNASer
tRNA charging by Aminoacyl-tRNA
synthetases is specific
2010-5-14
38
Class I,attach the amino acids to the
2’OH of the tRNA,and is usually
monomeric.
Class II,attach the amino acids to
the 3’OH of the tRNA,and is usually
dimeric or tetrameric
There are two classes of tRNA
synthetases.
2010-5-14
39
3-4 tRNA synthetases
recognize unique structure
features of cognate tRNAs
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
? The recognition has to ensure two
levels of accuracy,(1) each tRNA
synthetase must recognize the
correct set of tRNAs for a
particular amino acids; (2) each
synthetase must charge all of
these isoaccepting tRNAs (即由一种
synthetase所识别的不同 tRNAs)
2010-5-14
40
? The specificity determinants
for accurate recognition are
clusters at two distinct sites,
the acceptor stem and the anti-
codon loop.
2010-5-14
41
Fig 14-8
Fig 14-7
2010-5-14
42
3-5 Aminoacyl-tRNA
formation is very accurate,
selection of the correct
amino acid
? The aminoacyl tRNA synthetases
discriminate different amino acids
according to different natures of their
side-chain groups.
? Some enzymes have editing pocket
to do proofreading by matching the
wrong product and hydrolyzing it (3-
6).
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
43
Ribosome is responsible to
place the charged tRNAs onto
mRNA through base pairing of
the codon in mRNA and
anticodon in tRNA
3-7 Ribosomes is unable to
discriminate between
correctly or incorrectly
charged tRNAs (是否携带正确
的氨基酸 )
AT
TA
CHME
NT
O
F
AMI
NO
A
CI
DS
T
O
tR
NA
2010-5-14
44
Topic 4,the ribosome
Fig 14-17 two views of the ribosome
2010-5-14
45
4-1 the ribosome is composed
of a large and a small subunit
? The large subunit contains the
peptidyl transferase center,which is
responsible for the formation of
peptide bonds.
? The small subunit interacting with
mRNA contains the decoding center,
in which charged tRNAs read or
“decode” the codon units of the
mRNA.
RI
BO
SO
ME
S
2010-5-14
46
Fig 14-13** Ribosome
2010-5-14
47
4-2,the large and the small
subunits undergone
association and dissociation
during each cycle of
translation
RI
BO
SO
ME
S
2010-5-14
48
?Ribosome cycles,In cells,the
small and large ribosome subunits
associate with each other and the
mRNA,translate it,and then
dissociate after each round of
translation,This sequence of
association and dissociation is
called the ribosome cycle.
2010-5-14
49
Fig 14-14 Overview of the events of
translation/ribosome cycle
2010-5-14
50
Polysome/polyribosome,an
mRNA bearing multiple ribosomes
? Each mRNA can be translated
simultaneously by multiple ribosomes
Fig 14-15 A polyribosome
2010-5-14
51
RI
BO
SO
ME
S
4-3 New amino acids are
attached to the C-terminus of
the growing polypeptide
chain.
Protein is synthesized in a N-
to C- terminal direction
4-4 Peptide bonds are formed
by transfer of the growing
peptide chain from peptidyl-
tRNA to aminoacyl-tRNA.
2010-5-14
52
Fig 14-16
2010-5-14
53
The structure of the ribosome
4-5 Ribosomal RNAs are both
structural and catalytic
determinants of the ribosomes
4-6 The ribosome has three
binding site for tRNA.
4-7 Channels through the
ribosome allow the mRNA and
growing polypeptide to enter
and/or exit the ribosome.
RI
BO
SO
ME
S
2010-5-14
54F
ig 1
4-
19
3
-D
st
ru
ct
ur
e
of
th
e
ribo
so
me
inc
ludi
ng
3 bo
un
d
tR
NA
2010-5-14
55
Three binding site for tRNAs
Fig 14-18 A site,to bind the
aminoacylated-tRNA
B-site,to bind the
peptidyl-tRNA
E-site,to bind the
uncharged tRNA
2010-5-14
56
Channels for mRNA entering and
exiting are located in the small
subunit (see Fig,14-18)
There is a
pronounced kink in
the mRNA between
the two codons at P
and A sites,This kink
places the vacant A
site codon for
aminoacyl-tRNA
interaction,
Fig 14-20
2010-5-14
57
Channel for polypeptide chain
exiting locates in the large
subunit (see Fig,14-18)
The size of the
channel only
allow a very
limited folding
of the newly
synthesized
polypeptide
Fig 14-21
2010-5-14
58
? We now know that the rRNAs are not
simply structural components of the
ribosomes,Rather,they account for the
key function of the ribosomes,
? Most ribosomal proteins are on the
periphery of the ribosomes,not in its
interior,
? So,it’s inferred that the contemporary
ribosome evolved from a primitive
protein synthesis machine that was
composed entirely of RNA.
2010-5-14
59
Translation process
T5,Initiation of translation
T6,Elongation of translation
T7,termination of translation
Watch the animation
on your study CD
2010-5-14
60
Questions
1,Compare the mechanism of
translation initiation in prokaryotes
and eukaryotes (similarity and
difference)
2,How do prokaryotes and eukaryotes
find the translation start sites?
3,How do aminoacyl-tRNA
synthetases and the ribosomes
contribute to the fidelity of
translation,respectively?
2010-5-14
61
Overview of the events of
translation
Termination Elongation
Initiation
Fig 14-14
2010-5-14
62
5-6 Translation initiation factors
hold eukaryotic mRNAs in circles
Try to explain how the mRNA poly-A tail
contributes to the translation efficiency?
INI
TI
AT
IO
N
OF
T
RAN
SL
AT
IO
N
Fig 14-29
2010-5-14
63
6-3 Ribosome is a ribozyme
EL
ONG
AT
IO
N
OF
T
RAN
SL
AT
IO
N
Fig 14-33
Catalysis requires distance in the 1-3 ?,
and only RNA residues are present 18 ?
from the active site.
2010-5-14
64
EF-G mimics a tRNA molecule so as to
displace the tRNA bound to the A site
EF-G-GDPEF-Tu-GDPNP-Phe-tRNA
Fig 14-35
2010-5-14
65
Topic 8,translation dependent
regulation of mRNA and
protein stability
?Here regulation refers
cellular processes that deal
with defective mRNA and
their translated product.
2010-5-14
66
8-1,The SsrA RNA rescues (拯救 )
ribosomes that translate broken
mRNAs lacking a stop codon
(prokaryotes)
1.The ribosomes are trapped or
stalled on the broken mRNA
lacking a stop codon
2.The stalled ribosomes are
rescued by the action of a
chimeric RNA molecule that is
part tRNA and part mRNA,called
tmRNA.
3.SsrA is a 457-nt tmRNA
2010-5-14
67
Fig 14-39 SsrA rescues the stalled ribosomes
2010-5-14
68
8-2,Eukaryotic cells degrade
mRNAs that are incomplete or
have premature stop codons
Translation is tightly linked to the
process of mRNA decay in
eukaryotic cells
2010-5-14
69
When an mRNA contains a premature
stop codon (nonsense codon),the
mRNA is rapidly degraded by
nonsense mediated mRNA decay.
Pre-releasing the ribosome at the
nonsense codon prior to reaching the
exon-junction complex initiates a talk
between the complex and ribosome
to remove the 5’ cap from the mRNA
Nonsense mediated mRNA decay
2010-5-14
70
1,Translation of a normal
eukaryotic mRNA displace all
the exon junction complex
Fig 14-40a
2010-5-14
71
2,Nonsense mediated mRNA
decay
Fig 14-40b
2010-5-14
72
Non-stop mediated mRNA decay
rescues ribosomes that translate
mRNAs lacking a stop codon
(1)The lack of a stop codon results in
ribosome translation into the poly-A tail
to produce poly-Lys at the C-terminus
of the polypeptide; the poly-Lys marks
the newly synthesized for rapid
degradation,
Nonstop mediated decay
2010-5-14
73Fig 14-40c
(2)The ribosome eventually stalls at the 3’ end of
the mRNA,which is bound by the Ski7 protein
that triggers the ribosome dissociation and
recruits a 3’-5’ exonuclease activity to degrade
the,nonstop” mRNA,
2010-5-14
74
1,The main challenge of translation and
the solution
2,The structure and function of four
components of the translation
machinery.
3,Translation initiation,elongation and
termination (具体过程和翻译因子的作用 )
4,The mRNA and protein stability
dependent on translation (生物学问题是
什么，怎么解决的 )
Key points of the chapter
