Reading frame15′- HUU CU GGA CC U GG A GA U UC A CIA GU-8 Reading frame2--UU C UC G GA C CUG GAGAUU CA C AGU Reading frame3--UUCUCGGACCUGGAGA A GU Each mRNA molecule would have three potential reading frames(but only one usually codes for a polypeptide chain)
Each mRNA molecule would have three potential reading frames (but only one usually codes for a polypeptide chain)
4. The genetic codes were deciphered by simply using the in vitro protein synthesis system Artificially synthesized poly(U), synthesized using polynucleotide phosphorylase, was added to 20 reaction tubes each containing the cell-free E coli extract, GTP, atp a mixture of 20 amino acids and one 14C labeled amino acid Radioactive polypeptide was only detected in the tube containing [4C]-Phe(with high concentration of Mg2+t When poly(a)and poly(c) were added, radioactive polypeptides were only detected in the tubes containing 14C-labeled L-Lys and L-Pro respectively
4. The genetic codes were deciphered by simply using the in vitro protein synthesis system • Artificially synthesized poly(U), synthesized using polynucleotide phosphorylase, was added to 20 reaction tubes each containing the cell-free E.coli extract, GTP, ATP, a mixture of 20 amino acids, and one 14C labeled amino acid. • Radioactive polypeptide was only detected in the tube containing [14C]-Phe (with high concentration of Mg2+ ). • When poly (A) and poly (C) were added, radioactive polypeptides were only detected in the tubes containing 14C-labeled L-Lys and L-Pro respectively
UUU, AAA, CCC encodes Phe, Lys, Pro respectively. When poly(g) was added, no polypeptides synthesized prabably due to the formation of tetraplexes of the poly(G)strands
• UUU, AAA, CCC encodes Phe, Lys, Pro respectively. • When poly(G) was added, no polypeptides synthesized prabably due to the formation of tetraplexes of the poly (G) strands
5. Base composition of the triplets coding for some amino acids were revealed using mixed copolymers of rNa The composition of an RNa synthesized using polyribonucleotide phosphory lase depends on the proportion of each NDP present in the reaction mixture Investigation of the identity and quantity of the amino acids incorporated into the polypeptides in response to random polymers of rNa made from various ratios of NDPs can reveal the nucleotide composition(but not exact sequence) of the triplets corresponding to certain amino acids
5. Base composition of the triplets coding for some amino acids were revealed using mixed copolymers of RNA • The composition of an RNA synthesized using polyribonucleotide phosphorylase depends on the proportion of each NDP present in the reaction mixture. • Investigation of the identity and quantity of the amino acids incorporated into the polypeptides in response to random polymers of RNA made from various ratios of NDPs can reveal the nucleotide composition (but not exact sequence) of the triplets corresponding to certain amino acids
Table 15-1 Amino Acid Incorporation into Proteins Sum of Observed Tentative Calculated Triplet Frequency Calculated Amino acid Codon Triplet Amino acid Incorporation Assignments 3A 2AIC 1A2C 30 Frequencies Poly ac (5: 1) asparagine 24 2AIC Glutamine 24 2AIC Histidine 1A2C 4.0 Lysine 100 3A 100 Proline 1A2C, 3C 4.0 0.8 4.8 Threonine 2AIC. 1A20 4.0 Poly AC (1: 5) Asparagine 2AIC 3.3 Glutamine 5 2AIC 3.3 Histidine 1A2C 16.7 16.7 Lysine 1 3A 0.7 0.7 Proline 1A2C, 3C 16.7 83.3 100 Threonine 21 2AIC, 1A2C 3.3 16.7 20 The amino acid incorporation into proteins was observed after adding random copolymers of A and C to a cell-free extract similar to that described in Figure 15-1. The incorporation is given as a percentage of the maximal incorporation of a single amino acid. The copolymer ratio was then used to calculate the frequency with which a given codon would appear in the polynucleotide product. The relative frequencies of the codons are a function of the probability that a particular nucleotide will occur in a given position of a codon. For example, when the A/C ratio is 5: 1. the ratio of AAA/AAC=5x5x 5: 5 X5x 1=125: 25. If we thus assign to the 3A codon a frequency of 100, then the 2A and 1c codon is assigned a frequency of 20. By correlating the relative frequencies of amino acid incorporation with the calculated frequencies with which given codons appear, tentative codon assignments can be made
Composition of triplets coding for certain amino acids were assigned