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I4“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
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 (NMR)n nPi
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
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 gl
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 + mCDP (AMP)n(CMP)m+n+m)Pi e.g. ADP: CDP (AMP)5-(CMP)1 5:1 e.g. AACAAAAACAACAAAAAAAAACAA
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 + mCDP + (n+m)Pi 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 of1/6 Possible trinucleotides(codons )and resulting amino acids incorporated during translation Compo- Num Relative Amino Frea sition ber Frequency Freq.Acid of incorp. 3A 0.5781.0 LVS 1.0 2A,1C33x×0.1163x0.20Thr*,Asn,Gn0.27*,0.24,0.24 1A,2C33×0.0233X0.04Pr*,His,Thr*0.07,0.07,(04) 3C 0.0050.01 Pro (0.01*)
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 NNN NNN 大 AA-tRNA ribosome Binds to filter Which trinucleotide will allow binding of a particular AA-tRNA to ribosomes? pmoles AA-tRNA bound with AA-tRNA no NNN UUU AAA CCC Phe-tRNA 0.34 560200.30 ys-tRNA0.800.56613」060 Pro-tRNA0240200.18073
Defined trinucleotides stimulate binding of particular aminoacyl-tRNAs to ribosomes + *AA-tRNA + ribosome NNN *AA-tRNA NNN 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: