Breaking the genetic code required a cellfree proteinsynthesizing system

of the newly synthesized protein could then be determined, and its sequence could be compared with that of the RNA. Unfortunately, there was no way at that time to determine the nucleotide sequence of a piece of RNA; so indirect methods were necessary to break the code.

The first clues to the genetic code came in 1961, from the work of Marshall Nirenberg and Johann Heinrich Matthaei. These investigators created synthetic RNAs by using an enzyme called polynucleotide phosphorylase. Unlike RNA polymerase, polynucleotide phosphorylase does not require a template; it randomly links together any RNA nucleotides that happen to be available. The first synthetic mRNAs used by Nirenberg and Matthaei were homopolymers, RNA molecules consisting of a single type of nucleotide. For example, by adding polynucleotide phos-phorylase to a solution of uracil nucleotides, they generated RNA molecules that consisted entirely of uracil nucleotides and thus contained only UUU codons (< Figure 15.9). These poly(U) RNAs were then added to 20 tubes, each containing a cell-free protein-synthesizing system and the 20 different amino acids, one of which was radioactively labeled. Translation took place in all 20 tubes, but radioactive protein appeared in only one of the tubes—the one containing labeled phenylalanine (see Figure 15.9). This result showed that the codon UUU specifies the amino acid phenylalanine. The results of similar experiments using poly(C) and poly(A) RNA demonstrated that CCC codes for proline and AAA codes for lysine; for technical reasons, the results from poly(G) were uninterpretable.

To gain information about additional codons, Nirenberg and his colleagues created synthetic RNAs containing two or three different bases. Because polynucleotide phosphory-lase incorporates nucleotides randomly, these RNAs contained random mixtures of the bases and are thus called random copolymers. For example, when adenine and cyto-sine nucleotides are mixed with polynucleotide phospho-rylase, the RNA molecules produced have eight different codons: AAA, AAC, ACC, ACA, CAA, CCA, CAC, and CCC. In cell-free protein-synthesizing systems, these poly(AC) RNAs produced proteins containing six different amino acids: asparagine, glutamine, histidine, lysine, proline, and threonine.

The proportions of the different amino acids in the proteins depended on the ratio of the two nucleotides used in creating the synthetic mRNA, and the theoretical probability of finding a particular codon could be calculated from the ratios of the bases. If a 4:1 ratio of C to A were used in making the RNA, then the probability of C occurring at any given position in a codon is 4/5 and the probability of A being in it is 1/5. With random incorporation of bases, the probability of any one of the codons with two Cs and one A (CCA, CAC, or ACC) should be 4/5 X 4/5 X % = 16/125 = 0.13, or 13%, and the probability of any codon with two As and one C (AAC, ACA, or CAA) should be / X / X 4/5 = 4/125 = 0.032, or about 3%. Therefore, an amino acid encoded by

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