三联密码的证实
1961年 Crick和 Brenner.S等证实了三联密码的真实性 。
他们用 T4染色体上的一个基因 （ rⅡ 位点 ） 通过用 原黄素 （ proflavin） 处理,可以使 DNA脱落或插入单个碱基,插入叫,加字,突变,脱落叫,减字,突变,无论加字和减字都可以引起 移码突变 。
Crick小组用这种方法获得一系列的 T4“加字,
和,减字,突变,再进行杂交来获得加入或减少一个,二个,三个的不同碱基数的系列突变 。
Tools available
Cell-free systems for translation
– From bacteria,plants and animals
Ability to synthesize polyribonucleotides
– Polynucleotide phosphorylase can make RNAs from
NDPs
– Physiological function,reverse reaction for RNA
degradation
nNDP (NMP)n nPi+
Homopolymers of RNA direct
synthesis of homopolypeptides
Use of a single NDP as a substrate for
polynucleotide phosphorylase will produce
a homopolymer of that nucletide
– UDP as a substrate results in polyU as the
product
Addition of polyU to a cell-free translation
system results in polyphenylalanine as the
product.
Results of using homopolymers
to program translation
UUU encodes Phe
AAA encodes Lys
CCC encodes Pro
GGG encodes Gly
Mixed co-polymers of RNA direct the
incorporation of particular amino acids
into polypeptides
Mix two NDPs in a known ratio,and polynucleotide
phosphorylase will catalyze synthesis of an RNA with
those 2 nucleotides in that ratio,but random order.
nADP (AMP)n-(CMP)m (n+m)Pi++ mCDP
e.g,ADP:CDP
5:1 (AMP)5-(CMP)1
e.g,AACAAAAACAACAAAAAAAAACAA
Frequency of incorporation of each amino acid
correlates with frequency of occurrence of a
particular trinucleotide
For a 5:1 ratio of ADP:CDP -> poly(AC) (5:1),A is
present in the RNA 5/6 of the time,and C is present at a
frequency of 1/6.
Possible trinucleotides (codons) and resulting amino acids
incorporated during translation:
Compo- Num- Relative Amino Freq,
sition ber Frequency Freq,Acid of incorp.
3A 1 0.578 1.0 Lys 1.0
2A,1C 3 3x0.116 3x0.20 Thr*,Asn,Gln 0.27*,0.24,0.24
1A,2C 3 3x0.023 3x0.04 Pro**,His,Thr* 0.07,0.07,(.04*)
3C 1 0.005 0.01 Pro** (0.01**)
Defined trinucleotides stimulate binding of
particular aminoacyl-tRNAs to ribosomes
+ *AA-tRNA +
ribosome
NNN*
AA-tRNANNN
Binds to filter
Which trinucleotide will allow binding of a particular AA-tRNA
to ribosomes?
AA-tRNA no NNN UUU AAA CCC
Phe-tRNA 0.34 1.56 0.20 0.30
Lys-tRNA 0.80 0.56 6.13 0.60
Pro-tRNA 0.24 0.20 0.18 0.73
pmoles AA-tRNA bound with:
1961年 Crick和 Brenner.S等证实了三联密码的真实性 。
他们用 T4染色体上的一个基因 （ rⅡ 位点 ） 通过用 原黄素 （ proflavin） 处理,可以使 DNA脱落或插入单个碱基,插入叫,加字,突变,脱落叫,减字,突变,无论加字和减字都可以引起 移码突变 。
Crick小组用这种方法获得一系列的 T4“加字,
和,减字,突变,再进行杂交来获得加入或减少一个,二个,三个的不同碱基数的系列突变 。
Tools available
Cell-free systems for translation
– From bacteria,plants and animals
Ability to synthesize polyribonucleotides
– Polynucleotide phosphorylase can make RNAs from
NDPs
– Physiological function,reverse reaction for RNA
degradation
nNDP (NMP)n nPi+
Homopolymers of RNA direct
synthesis of homopolypeptides
Use of a single NDP as a substrate for
polynucleotide phosphorylase will produce
a homopolymer of that nucletide
– UDP as a substrate results in polyU as the
product
Addition of polyU to a cell-free translation
system results in polyphenylalanine as the
product.
Results of using homopolymers
to program translation
UUU encodes Phe
AAA encodes Lys
CCC encodes Pro
GGG encodes Gly
Mixed co-polymers of RNA direct the
incorporation of particular amino acids
into polypeptides
Mix two NDPs in a known ratio,and polynucleotide
phosphorylase will catalyze synthesis of an RNA with
those 2 nucleotides in that ratio,but random order.
nADP (AMP)n-(CMP)m (n+m)Pi++ mCDP
e.g,ADP:CDP
5:1 (AMP)5-(CMP)1
e.g,AACAAAAACAACAAAAAAAAACAA
Frequency of incorporation of each amino acid
correlates with frequency of occurrence of a
particular trinucleotide
For a 5:1 ratio of ADP:CDP -> poly(AC) (5:1),A is
present in the RNA 5/6 of the time,and C is present at a
frequency of 1/6.
Possible trinucleotides (codons) and resulting amino acids
incorporated during translation:
Compo- Num- Relative Amino Freq,
sition ber Frequency Freq,Acid of incorp.
3A 1 0.578 1.0 Lys 1.0
2A,1C 3 3x0.116 3x0.20 Thr*,Asn,Gln 0.27*,0.24,0.24
1A,2C 3 3x0.023 3x0.04 Pro**,His,Thr* 0.07,0.07,(.04*)
3C 1 0.005 0.01 Pro** (0.01**)
Defined trinucleotides stimulate binding of
particular aminoacyl-tRNAs to ribosomes
+ *AA-tRNA +
ribosome
NNN*
AA-tRNANNN
Binds to filter
Which trinucleotide will allow binding of a particular AA-tRNA
to ribosomes?
AA-tRNA no NNN UUU AAA CCC
Phe-tRNA 0.34 1.56 0.20 0.30
Lys-tRNA 0.80 0.56 6.13 0.60
Pro-tRNA 0.24 0.20 0.18 0.73
pmoles AA-tRNA bound with:
