Translation
翻译翻译是将 mRNA上的遗传信息转变为蛋白质序列的过程
Translation
Translation device
– Genetic code
– tRNA
– Ribosome
Translation mechanism
– Initiation
– Elongation
– Termination
ORFS 是指新测 DNA序列中，由计算机辨别出的可能的编码区，是从起始密码子到终止密码子的一段连续密码子区域。
也有人认为是 DNA或 RNA中一段意义不明的、连续的、不重叠的编码区，有起始密码子而无终止子。
Open reading frames ORFS
开放阅读框,读码框重叠基因 指一个基因的编码区和另一个基因的编码区部分或全部重叠
AUG UAAAUG UAA
在原核生物中，核糖体基因有第二个起始密码子，不需要从模板上分离即可开始翻译重叠基因。 Eukaryotes tend to make use of
alternative RNA processing.
Overlapping genes
重叠基因一,Genetic code遗传密码
Features of genetic code
遗传密码的特点
遗传密码是 三联体 密码
遗传密码 无逗号
遗传密码 不重迭
遗传密码具有 通用性 univeral
遗传密码的使用有 选择性
遗传密码具有 简并性 (degeneracy,synonyms)
密码子有 起始密码子 start codon和 终止密码子
stop codon
反密码子中的,摆动,(wobble）
1,3nucleotides encode a single amino acid
For 4 nucleotides to encode 20 amino acids,you
need a coding unit of at least 3:
– A coding unit of 2 nucleotides can only encode 16 amino
acids (4x4)
– A coding unit of 3 nucleotides can only encode 64 amino
acids (4x4x4)
Insertions or deletions of 1,2,4,5,etc nucleotides
cause a severe loss of function resulting from a
change in the reading frame.
But insertions or deletions of 3,6,9,etc have little
effect on the phenotype,because the reading frame is
not affected for most of the mRNA.
2,Degeneracy of the code
Degeneracy refers to the fact that almost all amino
acids are encoded by multiple codons.
Degeneracy is found primarily in the 3rd position
of the codon,i.e,the nucleotide in the 3rd position
can change without changing the amino acid
specified.
In some cases,the 1st position is also degenerate.
The degeneracy of the code
sense codons,61
start codon,1
stop codon:3
3,Codons for initiation of
translation
Major codon for initiation is AUG
Regardless of codon used,the first amino acid
incorporated in E,coli is formyl-Met.
For the 4288 genes identified in E,coli:
AUG is used for 3542 genes.
GUG is used for 612 genes.
UUG is used for 130 genes.
AUU is used for 1 gene.
CUG may be used for 1 gene.
Codons for termination of
translation
UAA (ochre),UAG (amber),UGA (opal)
For the genes identified in E,coli:
UAA is used for 2705 genes.
UGA is used for 1257 genes.
UAG is used for 326 genes.
4,Genetic code is univeral (almost)
All organisms so far examined use the code
as originally deduced
We can produce a human protein by using
E,coli over-expression system!
The standard genetic code is widespread but
not universal,e.g,in RNA derived from
mitochondrial DNA
硒代半胱氨酸
Alterations of the universal code
5,Differential codon usage
Some codons are used much more frequently than
others to encode a particular amino acid.
The pattern of codon usage varies between species
and even among tissues within a species.
Correlates with tRNA abundance.
Pattern of codon usage can be a predictor of level of
expression of a gene.
Preferred codon usage is a help in reverse genetics.
Differential codon usage
Codon usage database,
http://www.kazusa.or.jp/codon/
6,Three possible reading frames
Mol,Biol,Gene,Fig,14-1
Some phage DNA segments contain overlapping
genes in different reading frames (Fig,32-6)
Maximal use of the little DNA that they can pack
inside their capsids
摆动假说 (wobble hypothesis)是由 Crick.F（ 1966年 ） 提出的 。 即当 tRNA的反密码子与 mRNA的密码子配对时前两对严格遵守碱基互补配对法则,但第三对碱基有一定的自由度可以,摆动,。 摆动假说也称为 三中读二 （ 2 out of 3 reading） 。
7,Wobble of the anticodon
N u c l e o t i d e a t 5 ’ -e n d
o f a n t i c o d o n
N u c l e o t i d e a t 3 ’ -e n d
o f c o d o n
G Ca n p a i r w i t h U o r C
C Ca n p a i r w i t h G
A Ca n p a i r w i t h U
U Ca n p a i r w i t h A o r G
I ( i n o s i n e ) Ca n p a i r w i t h A,U o r C
三中读二一般可分为三种情况：
(1) 第１,２两个碱基形成６个氢键时,可三中读二 。
如 ＣＣ Ｘ,ＣＧ Ｘ,ＧＣ Ｘ 和 ＧＧ Ｘ 。
(2) 第 1,2两个碱基形成 4个氢键时,不可三中读二 。
如 AAX,ＡＵ Ｘ,ＵＡ X 和 ＵＵ Ｘ 。
(3) 第１,２两个碱基形成５个氢键时,
当第二个碱基为嘧啶时,可三中读二；
如 ＵＣ Ｘ,ＡＣ Ｘ,ＣＵ Ｘ 和 ＧＵ Ｘ 。
当第二个碱基为嘌呤时则不能三中读二,
如 ＣＡ Ｘ,ＧＡ Ｘ,ＵＧ Ｘ 和 ＡＧ X。
Wobble of the anticodon
Codon-anticodon
recognition involves
wobbling
Wobble in base pairing
allows G-U pairs to form
between the third base of
the codon and the first base
of the anticodon
Third bases have the least influence on
codon meanings
一共有 8个 密码子家族,每家族的 4个密码子的前 2个碱基是相同的，而且 4个密码子具有相同的意义，所以它们的第 3个碱基在识别氨基酸过程中不起作用。
一共有 7个 密码子对,只要在第 3个位置上出现的是嘧啶碱基，它们将表达相同的意义。
一共有 5个密码子对，只要在第 3个位置出现的是嘌呤碱基，它们所编码的那个氨基酸将不会改变。
只有在 3种情况下,在第 3个位置上出现特定的碱基才使密码子被授予了唯一的意义,AUG
（ 编码蛋氨酸 ）,UGG（ 编码色氨酸 ） 和 UGA
（ 终止密码 ） 。 因此 C和 U在第 3个碱基的位置上永远不会具有唯一的意义,而 A也永远不会表示一种唯一的氨基酸 。
tRNA contains
modified bases
that influence
its pairing
properties
Base-pairing wobble
Modification to inosine allows pairing with U,C,and A
Modification to
2-thiouridine
restricts pairing
to A alone
because only one
H-bond can form
with G
8,Effect of mutation突变的影响
Synonym codons have the same meaning in the
genetic code,Synonym tRNAs bear the same
amino acid and respond to the same codon.
同义密码子 家族意味着可将突变的影响降到最小密码子处的碱基突变可分为两类：
碱基转换 Transitions,碱基由嘌呤到嘌呤或嘧啶到嘧啶的突变碱基颠换 Transversions:由嘌呤到嘧啶或嘧啶到嘌呤的碱基突变
transition and transversion
对特定氨基酸残基的影响
Transition Transversion
Third position no effect,over half no effect
except Met,Trp
Second position effect on AA effect on both
species only AA species and type
first position a similar type of AA,in few cases,no effect
on AA species like Leu,Arg.
9,Types of mutations in coding regions
Silent (synonymous)
– Do not change the encoded amino acid
– Occur in degenerate positions in the codon
– Are often not subject to purifying selection and thus
occur more frequently in evolution
Nonsilent (nonsynonymous)
– Do change the encoded amino acid
– Occur in non-degenerate positions in the codon
– Are more likely to be subject to purifying selection
and thus occur less frequently in evolution
Changes that alter the encoded
product
Missense,cause a replacement of an amino acid
– e.g,CAA (Gln) -----> CGA (Arg)
Nonsense,cause a termination of translation
– e.g,CAA (Gln) -----> UAA (term)
Frameshift,insertion or deletion that changes the
reading frame
– e.g,CAA (Gln) -----> C-A (frameshift)
Suppressor mutations
Mutations at a second site that can overcome the
effects of a missense or nonsense mutation are
suppressors.
Can be in the same gene (but affecting a different
codon) OR can be in a different gene.
Isolation of suppressor mutations in other genes
indicates that the product of the other gene
interacts with the product encoded by the gene
with the original mutation.
Nonsense Suppression
Amber (UAG),ochre (UAA),opal (UGA)
tRNA mutation recognizes a nonsense (stop)
codon
How do cells tolerates a mutation that both
elimination of normal tRNA and prevents
the termination of protein synthesis?
Mutated tRNA is usually minor member of a
set of tRNAs
Many mRNA have two tandem stop codons
二,tRNA的结构和功能
Primary structure of tRNA
Linear sequence,76nt (60~95nt)
Modified bases created post-transcriptionally,
accounting for 20% around of the total bases
Four of these are very common:
T ( thymidine ),? ( pseuouridine ),
dihydrouridine ( D ) and I ( inosine )
tRNA的 三叶草型 的二维结构
各种 tRNA中 22个碱基是恒定的
5’端和 3’端配对（常为 7bp） 形成茎区，称为 受体臂
（ acceptor arm） 或称 氨基酸臂 。在 3’端永远是 4个碱基
（ XCCA） 的单链区，在其末端有 2’ -OH或 3’ -OH,是被氨基酰化位点。此臂负责携带特异的氨基酸。
TψC常由 5bp的茎和 7Nt和环组成 。 此臂负责和核糖体上的 rRNA 识别结合
反密码子臂 (anticodon arm)常由 5bp的茎区和 7nt的环区组成，它负责对密码子的识别与配对
D环 (D arm)的茎区长度常为 4bp,也称 双氢尿嘧啶环。
负责和氨基酰 tRNA聚合酶结合
额外环 (extra arm)可变性大,从 4 nt到 21nt不等,其功能是在 tRNA的 L型三维结构中负责连接两个区域 （ D环 -反密码子环和 TψC-受体臂 ）
tRNA secondary
structure
tRNA tertiary structure
L-structure
在 tRNA的三维结构中 D环和 TψC环形成了 L型的转角 。
氨基酸受体臂位于 L型的一侧,距反密码子环约 70 A
在一些保守和半保守的碱基之间形成很多的三级氢键,使分子形成 L形,并使结构稳定 。
使得三维结构得以形成的这些碱基配对涉及到与磷酸核糖主链相互作用的三级结构的磷酸二酯键分布在核糖的 2’ -OH上 。
几乎所有的碱基平面之间产生堆积的作用 。
在反密码子茎中仅有很少的三级氢键
tRNA tertiary structure
tRNA tertiary structure
酵母苯丙氨酸 tRNA的三级氢键
tRNA三维结构中共有 9个三级氢键，这些氢键主要涉及几个恒定碱基之间的碱基配对，有助于稳定结构
tRNA的碱基堆积
Aminoacylation of tRNAs
tRNA分子氨基酰化
(1),AA activation
AA + ATP + E* Aminoacyl -AMP-E + PPi
E,aminonacyl-tRNA synthetase (ARS,氨酰 tRNA合成酶 )
(2),The appropriate tRNA replaces the AMP
Aminoacyl-AMP-E + tRNA AA - tRNA + AMP +E
(3),Some ARS can carry out a proofreading step.
氨酰 tRNA合成酶 ARS
The computer-generated models of GlnRS and
AspRS
Four different classes,2?4?2?2;
The length of polypeptide chains range from 334 to
1000AA;
They can be distinguished between 40 similarly shaped,
but different,tRNA molecules in cell,
They use,Anticodon
Identity elements (识别元件 ) of tRNAs,?
The various ARS enzyme can be quite
different
tRNA对氨基酸的识别
1988年 Hou Ya-ming（ 候雅明）和 Schimmel
用点突变的方法来改变校正 tRNA（ Ala）
上的各个位点，观察对识别 Ala有何影响。
他们发现了 Ala tRNA的 G3:U70碱基对，仅一对碱基决定了丙氨酰 tRNA合成酶与
tRNA的识别。这种识别位点称为 tRNA的
Identity elements 识别元件,或称为 副密码子
（ paracodon）。
tRNA对氨基酸的识别
Both anticodon and
identity elements in
tRNA are involved
in recognition by
ARS
氨酰 tRNA合成酶的鉴别功能
动力学校对 Kinetic profreading
化学校对 Chemical proofreading
动力学校对 Kinetic profreading
化学校对
Chemical
proofreading
翻译翻译是将 mRNA上的遗传信息转变为蛋白质序列的过程
Translation
Translation device
– Genetic code
– tRNA
– Ribosome
Translation mechanism
– Initiation
– Elongation
– Termination
ORFS 是指新测 DNA序列中，由计算机辨别出的可能的编码区，是从起始密码子到终止密码子的一段连续密码子区域。
也有人认为是 DNA或 RNA中一段意义不明的、连续的、不重叠的编码区，有起始密码子而无终止子。
Open reading frames ORFS
开放阅读框,读码框重叠基因 指一个基因的编码区和另一个基因的编码区部分或全部重叠
AUG UAAAUG UAA
在原核生物中，核糖体基因有第二个起始密码子，不需要从模板上分离即可开始翻译重叠基因。 Eukaryotes tend to make use of
alternative RNA processing.
Overlapping genes
重叠基因一,Genetic code遗传密码
Features of genetic code
遗传密码的特点
遗传密码是 三联体 密码
遗传密码 无逗号
遗传密码 不重迭
遗传密码具有 通用性 univeral
遗传密码的使用有 选择性
遗传密码具有 简并性 (degeneracy,synonyms)
密码子有 起始密码子 start codon和 终止密码子
stop codon
反密码子中的,摆动,(wobble）
1,3nucleotides encode a single amino acid
For 4 nucleotides to encode 20 amino acids,you
need a coding unit of at least 3:
– A coding unit of 2 nucleotides can only encode 16 amino
acids (4x4)
– A coding unit of 3 nucleotides can only encode 64 amino
acids (4x4x4)
Insertions or deletions of 1,2,4,5,etc nucleotides
cause a severe loss of function resulting from a
change in the reading frame.
But insertions or deletions of 3,6,9,etc have little
effect on the phenotype,because the reading frame is
not affected for most of the mRNA.
2,Degeneracy of the code
Degeneracy refers to the fact that almost all amino
acids are encoded by multiple codons.
Degeneracy is found primarily in the 3rd position
of the codon,i.e,the nucleotide in the 3rd position
can change without changing the amino acid
specified.
In some cases,the 1st position is also degenerate.
The degeneracy of the code
sense codons,61
start codon,1
stop codon:3
3,Codons for initiation of
translation
Major codon for initiation is AUG
Regardless of codon used,the first amino acid
incorporated in E,coli is formyl-Met.
For the 4288 genes identified in E,coli:
AUG is used for 3542 genes.
GUG is used for 612 genes.
UUG is used for 130 genes.
AUU is used for 1 gene.
CUG may be used for 1 gene.
Codons for termination of
translation
UAA (ochre),UAG (amber),UGA (opal)
For the genes identified in E,coli:
UAA is used for 2705 genes.
UGA is used for 1257 genes.
UAG is used for 326 genes.
4,Genetic code is univeral (almost)
All organisms so far examined use the code
as originally deduced
We can produce a human protein by using
E,coli over-expression system!
The standard genetic code is widespread but
not universal,e.g,in RNA derived from
mitochondrial DNA
硒代半胱氨酸
Alterations of the universal code
5,Differential codon usage
Some codons are used much more frequently than
others to encode a particular amino acid.
The pattern of codon usage varies between species
and even among tissues within a species.
Correlates with tRNA abundance.
Pattern of codon usage can be a predictor of level of
expression of a gene.
Preferred codon usage is a help in reverse genetics.
Differential codon usage
Codon usage database,
http://www.kazusa.or.jp/codon/
6,Three possible reading frames
Mol,Biol,Gene,Fig,14-1
Some phage DNA segments contain overlapping
genes in different reading frames (Fig,32-6)
Maximal use of the little DNA that they can pack
inside their capsids
摆动假说 (wobble hypothesis)是由 Crick.F（ 1966年 ） 提出的 。 即当 tRNA的反密码子与 mRNA的密码子配对时前两对严格遵守碱基互补配对法则,但第三对碱基有一定的自由度可以,摆动,。 摆动假说也称为 三中读二 （ 2 out of 3 reading） 。
7,Wobble of the anticodon
N u c l e o t i d e a t 5 ’ -e n d
o f a n t i c o d o n
N u c l e o t i d e a t 3 ’ -e n d
o f c o d o n
G Ca n p a i r w i t h U o r C
C Ca n p a i r w i t h G
A Ca n p a i r w i t h U
U Ca n p a i r w i t h A o r G
I ( i n o s i n e ) Ca n p a i r w i t h A,U o r C
三中读二一般可分为三种情况：
(1) 第１,２两个碱基形成６个氢键时,可三中读二 。
如 ＣＣ Ｘ,ＣＧ Ｘ,ＧＣ Ｘ 和 ＧＧ Ｘ 。
(2) 第 1,2两个碱基形成 4个氢键时,不可三中读二 。
如 AAX,ＡＵ Ｘ,ＵＡ X 和 ＵＵ Ｘ 。
(3) 第１,２两个碱基形成５个氢键时,
当第二个碱基为嘧啶时,可三中读二；
如 ＵＣ Ｘ,ＡＣ Ｘ,ＣＵ Ｘ 和 ＧＵ Ｘ 。
当第二个碱基为嘌呤时则不能三中读二,
如 ＣＡ Ｘ,ＧＡ Ｘ,ＵＧ Ｘ 和 ＡＧ X。
Wobble of the anticodon
Codon-anticodon
recognition involves
wobbling
Wobble in base pairing
allows G-U pairs to form
between the third base of
the codon and the first base
of the anticodon
Third bases have the least influence on
codon meanings
一共有 8个 密码子家族,每家族的 4个密码子的前 2个碱基是相同的，而且 4个密码子具有相同的意义，所以它们的第 3个碱基在识别氨基酸过程中不起作用。
一共有 7个 密码子对,只要在第 3个位置上出现的是嘧啶碱基，它们将表达相同的意义。
一共有 5个密码子对，只要在第 3个位置出现的是嘌呤碱基，它们所编码的那个氨基酸将不会改变。
只有在 3种情况下,在第 3个位置上出现特定的碱基才使密码子被授予了唯一的意义,AUG
（ 编码蛋氨酸 ）,UGG（ 编码色氨酸 ） 和 UGA
（ 终止密码 ） 。 因此 C和 U在第 3个碱基的位置上永远不会具有唯一的意义,而 A也永远不会表示一种唯一的氨基酸 。
tRNA contains
modified bases
that influence
its pairing
properties
Base-pairing wobble
Modification to inosine allows pairing with U,C,and A
Modification to
2-thiouridine
restricts pairing
to A alone
because only one
H-bond can form
with G
8,Effect of mutation突变的影响
Synonym codons have the same meaning in the
genetic code,Synonym tRNAs bear the same
amino acid and respond to the same codon.
同义密码子 家族意味着可将突变的影响降到最小密码子处的碱基突变可分为两类：
碱基转换 Transitions,碱基由嘌呤到嘌呤或嘧啶到嘧啶的突变碱基颠换 Transversions:由嘌呤到嘧啶或嘧啶到嘌呤的碱基突变
transition and transversion
对特定氨基酸残基的影响
Transition Transversion
Third position no effect,over half no effect
except Met,Trp
Second position effect on AA effect on both
species only AA species and type
first position a similar type of AA,in few cases,no effect
on AA species like Leu,Arg.
9,Types of mutations in coding regions
Silent (synonymous)
– Do not change the encoded amino acid
– Occur in degenerate positions in the codon
– Are often not subject to purifying selection and thus
occur more frequently in evolution
Nonsilent (nonsynonymous)
– Do change the encoded amino acid
– Occur in non-degenerate positions in the codon
– Are more likely to be subject to purifying selection
and thus occur less frequently in evolution
Changes that alter the encoded
product
Missense,cause a replacement of an amino acid
– e.g,CAA (Gln) -----> CGA (Arg)
Nonsense,cause a termination of translation
– e.g,CAA (Gln) -----> UAA (term)
Frameshift,insertion or deletion that changes the
reading frame
– e.g,CAA (Gln) -----> C-A (frameshift)
Suppressor mutations
Mutations at a second site that can overcome the
effects of a missense or nonsense mutation are
suppressors.
Can be in the same gene (but affecting a different
codon) OR can be in a different gene.
Isolation of suppressor mutations in other genes
indicates that the product of the other gene
interacts with the product encoded by the gene
with the original mutation.
Nonsense Suppression
Amber (UAG),ochre (UAA),opal (UGA)
tRNA mutation recognizes a nonsense (stop)
codon
How do cells tolerates a mutation that both
elimination of normal tRNA and prevents
the termination of protein synthesis?
Mutated tRNA is usually minor member of a
set of tRNAs
Many mRNA have two tandem stop codons
二,tRNA的结构和功能
Primary structure of tRNA
Linear sequence,76nt (60~95nt)
Modified bases created post-transcriptionally,
accounting for 20% around of the total bases
Four of these are very common:
T ( thymidine ),? ( pseuouridine ),
dihydrouridine ( D ) and I ( inosine )
tRNA的 三叶草型 的二维结构
各种 tRNA中 22个碱基是恒定的
5’端和 3’端配对（常为 7bp） 形成茎区，称为 受体臂
（ acceptor arm） 或称 氨基酸臂 。在 3’端永远是 4个碱基
（ XCCA） 的单链区，在其末端有 2’ -OH或 3’ -OH,是被氨基酰化位点。此臂负责携带特异的氨基酸。
TψC常由 5bp的茎和 7Nt和环组成 。 此臂负责和核糖体上的 rRNA 识别结合
反密码子臂 (anticodon arm)常由 5bp的茎区和 7nt的环区组成，它负责对密码子的识别与配对
D环 (D arm)的茎区长度常为 4bp,也称 双氢尿嘧啶环。
负责和氨基酰 tRNA聚合酶结合
额外环 (extra arm)可变性大,从 4 nt到 21nt不等,其功能是在 tRNA的 L型三维结构中负责连接两个区域 （ D环 -反密码子环和 TψC-受体臂 ）
tRNA secondary
structure
tRNA tertiary structure
L-structure
在 tRNA的三维结构中 D环和 TψC环形成了 L型的转角 。
氨基酸受体臂位于 L型的一侧,距反密码子环约 70 A
在一些保守和半保守的碱基之间形成很多的三级氢键,使分子形成 L形,并使结构稳定 。
使得三维结构得以形成的这些碱基配对涉及到与磷酸核糖主链相互作用的三级结构的磷酸二酯键分布在核糖的 2’ -OH上 。
几乎所有的碱基平面之间产生堆积的作用 。
在反密码子茎中仅有很少的三级氢键
tRNA tertiary structure
tRNA tertiary structure
酵母苯丙氨酸 tRNA的三级氢键
tRNA三维结构中共有 9个三级氢键，这些氢键主要涉及几个恒定碱基之间的碱基配对，有助于稳定结构
tRNA的碱基堆积
Aminoacylation of tRNAs
tRNA分子氨基酰化
(1),AA activation
AA + ATP + E* Aminoacyl -AMP-E + PPi
E,aminonacyl-tRNA synthetase (ARS,氨酰 tRNA合成酶 )
(2),The appropriate tRNA replaces the AMP
Aminoacyl-AMP-E + tRNA AA - tRNA + AMP +E
(3),Some ARS can carry out a proofreading step.
氨酰 tRNA合成酶 ARS
The computer-generated models of GlnRS and
AspRS
Four different classes,2?4?2?2;
The length of polypeptide chains range from 334 to
1000AA;
They can be distinguished between 40 similarly shaped,
but different,tRNA molecules in cell,
They use,Anticodon
Identity elements (识别元件 ) of tRNAs,?
The various ARS enzyme can be quite
different
tRNA对氨基酸的识别
1988年 Hou Ya-ming（ 候雅明）和 Schimmel
用点突变的方法来改变校正 tRNA（ Ala）
上的各个位点，观察对识别 Ala有何影响。
他们发现了 Ala tRNA的 G3:U70碱基对，仅一对碱基决定了丙氨酰 tRNA合成酶与
tRNA的识别。这种识别位点称为 tRNA的
Identity elements 识别元件,或称为 副密码子
（ paracodon）。
tRNA对氨基酸的识别
Both anticodon and
identity elements in
tRNA are involved
in recognition by
ARS
氨酰 tRNA合成酶的鉴别功能
动力学校对 Kinetic profreading
化学校对 Chemical proofreading
动力学校对 Kinetic profreading
化学校对
Chemical
proofreading
