三联密码的证实.pptVIP

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 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. 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: Defined trinucleotides stimulate binding of particular aminoacyl-tRNAs to ribosomes Repeating sequence synthetic polynucleotides direct incorporation of particular amino acids * nNDP (NMP)n nPi + nADP (AMP)n-(CMP)m (n+m)Pi + + mCDP e.g. ADP:CDP 5:1 (AMP)5-(CMP)1 e.g. AACAAAAACAACAAAAAAAAACAA 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