Purpose of experiment:  To overcome the purely chemical problems of building short polynucleotide         chains and by a combination of methods of enzymology and chemistry to undertake a precise study of the genetic code...
 

Previous experiments discussed in background important in delvelopment of Khorana's work....

Procedure:  1) Chemical Synthesis of Polynucleotides of Defined Sequences...

                         2) Synthetic Deoxyribopolynucleotides As Templates For the Dna Polymerase of E. Coli
      - synthesis of high molecular weight polymers is extensive.... see results
       -  reactions showed exponential kinetics at 37 degrees and duration of the lag period appears to increase with decreasing       chain length of polynucleotides...
       - little or no lag observed only when short pieces of both complementary strands present...
       - products of extensive synthesis contain the apprpriate nucleotides in perfectly alternating sequences...
       - the high molecular weight product can be reutilized for more symthesis... ie. can skip initial synthesis steps....
 

3) Synthetic Deoxyribopolynucleotides as Templates for RNA Polymerase
      - use of polynucletides with repeating dinucleotide and trinucleotide seuences used...
       -  giving only the nucleoside triphosphates required for copying one strand restricts RNA polymerase action to that strand...as only 2-3 bases are needed...
       -  use of DNA polymerase and RNA polymerase had at disposal a variety of high molecular weight ribopolynucleotides of known sequences... mistake levels, if they occured at all, were insignificant...
 
 

4) Cell-Free Polypeptide Synthesis Using Polynucleotides With Repeating Sequences
      - use of di- and triribonucletides to synthesize proteins...
       - thus the genetic code could be successfully attcked on the basis of known sequences coding for specific amino acids....
       - used a cell-free system with artificially high Mg 2+ conc. which enabled polypeptide chains to start without a proper initiation signal... ie AUG not needed
 

5) Codon Assignments

       - Nirenberg and Leder(1964) binding technique...first event in synthesis of peptide bond is formation of a ternary complex between messenger RNA, the ribosome, and aminoacyl-tRNA
       - messenger RNA selects aminoacyl-tRNA from the soluble pool
       - Nirenberg and Leder's simplification was that the messenger may bo "often" as short as a trinucleotidein directing binding
       - one can therefore look for the stimulation of the binding of different aminoacyl-tRNA to ribosomes in the presence of specific trinucleotides...
       - use measures of C14-lysyl-tRNA binding to ribosomes in the presence of increasing amounts of specified trnucleotides

6) Codons Involved in Protein Chain Initiation in E. coli
     - widely believed that formylmethionine (fmet) as carried by formyl-met-tRNA is intiation signal(Marcker and Sanger 1964)
       - starts with fmet at amino terminus
       - poly r-AUG shown to direct polymethionine synthesis
       - also dicovered that GUG also a start codon....

7) Role of Ribosomal Subunits in Initiation and Protein Synthesis
     - 70s ribosome....
        - 30s and 50s roles in protein synthesis (Gosh and Khorana, 1967)
        - they act as two binding sites and play a major role in protein synthesis... results and conclusions clarify this role...
 

8) Transfer Ribonucleic Acids: The Anticodons and Codon Recognition
       - refered to "secondary structure" of tRNA... the cloverleaf (RajBhandary et al. 1967)
       - tRNA recognized as adapter.. an imprtant advance
       - tRNA contains anticodon loop which is composed of triplet of complementary bases to ssRNA
       - amino acid on end of tRNA molecule