Deciphering the Code

   How was the triplet nucleotide code discovered?

          It was known that there were 20 naturally occuring amino acids.
          If it was a 1- or 2- letter code then
                   a) a one letter code will result in 4 amino acids (A, C, G, T)
                   b) a two letter code will result in 16 amino acids (combinations of A, C, G, and T)
          Therefore to produce 20 amino acids a minimum of 3 letters is required. Hence a 3-letter code.

Experimental Asumptions

   1) Three letter-code for the known 20 amino acids
   2) 64 possible triplets coding for varoius amino acids
   3) mRNA coded using triplets
   4) Nonoverlapping property of the code
         - the nucleotide sequence for example ACGACGACG...
           is read as ACG ACG ... and not as ACG CGA GAC... (overlapping)
   5) Some sort of stop and start codon

Experimental Data and Results

Previous to Khorana's work on the genetic code, Nirenberg laid the foundation for deciphering the genetic code. He performed experiments using repeating sequences of the same nucleotide of mRNA and comparing it to the amino acid produced.

He found the following:

     Poly U -----  UUUUUUUUU...   produced poly-phenylalanine, so   UUUcodes for phe (phenyalanine)
    Poly A -----  AAAAAAAAA....  produced poly- lysine, so              AAA  codes for lys (lysine)
    Poly C -----  CCCCCCCC...    produced poly proline, so             CCC codes for pro (proline)
    Poly G -----  GGGGGGGG...    produced poly- glycine, so           GGG codes for gly (glycine)

Data:

   An example of di-, tri-, and tetra-nucleotide polymers (Fig.1).

   Khorana's experiment was based on different combinations of repeating nucleotide sequences of
   mRNA which he placed in a cell-free environment.

   Repeating sequences of two, three and four nucleotides combinations of DNA used in the experiment
   (Fig.2).

   These were then used to produce double stranded DNA molecules by combining them together, and
   transcribing the DNA molecules using RNA polymerase to produce single stranded poly mRNA
   molecules like poly AC mRNA (messenger RNA molecule with repeating AC sequence) (Refer to
   procedure).

   The results obtained in translating these polynucleotide mRNA as follows:
         a) Dinucleotide (Fig.3)
         b) Trinucleotide(Fig. 4)
         c) Tetranucleotide (Fig 5)

Codon Assignments:

   How were the nucleotide triplets assigned to each amino acid?

   There were two ways in which this was done:

   1) Using Logical Deductions:

        The different nucleotide sequences with the corresponding amino acids were compared and the
         triplet code for the amino acids was determined.

   Example:
      (Note: * denotes initiation sites)

        i) Comparison of :

    Poly AC (poly-dinucleotide)

         AC + AC+ AC + AC... ---> ACACACAC... ---> ACA-CAC-ACA-CAC...    mixture of poly-thr and
                                                                                   or CAC-ACA-CAC-ACA...    his

     Poly CAA(poly-trinucleotide)

          CAA + CAA + CAA... ---> C*A*A*CAACAACAA... ---> CAA-CAA-CAA...   mixture of
                                                                                              or AAC-AAC-AAC... poly-thr, gln and asn
                                                                                                     or ACA-ACA-ACA...

              Since ACA and thr occur in both experiments, then ACA makes thr (theronine).
              So CAC makes his (histidine). Therefore either AAC or CAA makes either
              gln (glutamine) or asn (asparagine). There was not enough data present in the
              original paper to further deduce which code belonged to which amino acid.

        ii) Comparison of:

    Poly UUC (poly-trinucleotide)

    UUC + UUC + UUC... ---> U*U*C*UUC... ---> UUC-UUC...      mixture of poly-phe, ser and leu
                                                                                  or UCU-UCU...
                                                                                  or CUU-CUU...

   Poly UUAC (poly-tetranucleotide)

   UUAC + UUAC... ---> U**U*A*C*UUAC.. ---> UUA-CUU-ACU-UAC...   mixture of poly-leu, leu,thr
                                                                                 or UAC-UUA-CUU-ACU...   and tyr
                                                                                 or ACU-UAC-UUA-CUU...
                                                                                 or CUU-AUC-UAC-UUA...

              CUU and leu are common to both experiments, hence CUU makes leu.
              Compare poly UUC to poly UC:

   Poly UC (poly-dinucleotide)

   UC + UC + UC... ---> UCUCUC...  ---> UCU-CUC...                mixture of poly-leu and ser
                                                                       or CUC-UCU...

              UCU and ser are common to each and hence UCU makes ser (serine).
              Therefore CUC makes leu (leucine) and  UUC makes phe (phenylalanine).

        iii) Comparison of the last two poly-tetranucleotides (Fig.5) indicates the formation of a
            mixture of di- and tri-peptides, indicating that there were nonsense triplets in the sequences.
            By using a similar sort of deduction as above UAA and UAG are seen as stop codes.

         Since leucine from the above two examples is coded by two triplets, CUC and CUU, Khorana
         concluded about the idea of degeneracy, where more than one triplet may code for a single
         amino acid. The idea of the wobble position and degeneracy was observed by Crick in 1966,
         when he developed the wobble hypothesis. Whether Khorana knew about the wobble position
         is questionable.

   2) Binding Assay

        Beacause more than one triplert may code for any single amino acid and a large number of
        polypepetide syntheses is required to use the method mentioned above, it is easier to match
        the possible triplets with the amino acids using an experimental method.

        Khorana used a technique developed by Nirenberg and Leder (see procedure).

        For example a poly-AAG sequence which produced a mixture of poly-lysine, glutamate, and
        arginine can use the binding technique (binding of aminoacyl-tRNA to ribosome in the presence
        of specific trinucleotides). .(Fig.6)

Conclusions:

   Codon assignments led to:
        (i) the assignment of 51 out of the 64  possible triplets to amino acids (Fig.7)

        (ii) the formation of a genetic code table (Fig 8)