SESSION IV. DISCUSSION 377 



We suggest that these anhydrides are typical specimens of these labile starting com- 

 pounds with high levels of free energy from which protein is synthesized. In this con- 

 nection I should like to deal shortly with some experiments which have been carried out 

 in our laboratory and are still in progress. 



In our first experiments in 1950 (with Nidzyan) it was shown that when isolating ribo- 

 nucleic acid from yeast or from animal tissues one can transfer to it the external phosphate 

 groups of ATP using a small quantity of a homogenate of Uver as a transferring enzyme. 

 These experiments have been repeated and confirmed by Brächet and also by Dounce 

 &Kay. 



The latter workers have shown that, for this transphosphorylation, one can use purified 

 myokinase, the well known enzyme which brings about the disproportionation of nucleo- 

 tide polyphosphates. Using this enzyme they were able to prepare phosphorylated RNA 

 in considerable quantities. In our work (with Rubina) a similar phosphorylation of RNA 

 (from yeast and pancreas) was obtained, the excess of phosphate amounting to as much 

 as 70%. This excess of phosphate could be hydrolysed by acid Uke the phosphate of ATP. 

 Our present views of the structure of RNA, put forward by Todd and his school, indicate 

 that the phosphoric acid in RNA is combined with the 5' position of the ribose, just as it 

 is in ATP, and the structure of each link in the polymer is just like the structure of adenyUc 

 acid. The completeness of the analogy between the behaviour of nucleotide phosphates of 

 low molecular weight and that of their polymeric analogues should therefore occasion no 

 surprise. 



The next step was to demonstrate the macroergic character of the excess phosphate in 

 phosphorylated RNA. This was done in two typical cases. The acceptor used at first was 

 fructose 6-phosphate which, with phosphorylated RNA and the ordinary enzjone phos- 

 phofructokinase, is converted into fructose diphosphate. This reaction had the advantage 

 that it permitted very specific enzymic methods of control to be applied. The product of 

 the reaction (more correctly its trichloroacetic acid filtrate) was treated with aldolase 

 which converted it into triose phosphates, substances which can easily be estimated 

 quantitatively. The reaction of transfer of phosphate groups from phosphorylated RNA 

 to fructose 6-phosphate went very well, just as well as with ATP itself. 



The other substances which we used as an acceptor for phosphate was creatine. By the 

 use of the enzyme creatine-phosphotransferase from muscle phosphate was transferred 

 from RNA to creatine, which demonstrated conclusively the macroergic nature of phos- 

 phorylated RNA. 



It should also be remembered that phosphate-rich preparations of RNA, containing 

 an excess of phosphate as compared with the ordinary formula, have been obtained by a 

 nimiber of authors (Brächet, Belozerskiî) from yeast and other cells and tissues in a state 

 of active growth. This also would seem to be an argument in favour of the idea that 

 phosphorylated RNA is not an artifact but is an important endogenous substance of cells. 



AU this enables us to put forward the hypothesis that, under the influence of such 

 enzymic systems as were used by Hoagland in his experiments, amino acid residues should 

 combine with phosphorylated RNA, forming anhydrides with the phosphate groups and, 

 at the same time, splitting off mineral phosphate. It seems to us very probable that such a 

 reaction occurs and it provides a means for the combination of amino acids with a tem- 

 plate in the form of a labile compound with a high level of free energy. The later hydro- 

 lysis of such anhydrides could be accompanied by their condensation into polypeptide 

 chains. Thermodynamically such a process is quite possible. It is shown to be chemically 

 possible by non-enzymic model experiments carried out by many authors actually using 

 phosphoric acid derivatives of amino acids (e.g. by the experiments of Katchalski). 



At present we are trying to prove that amino acids really are bound in the form of 

 anhydrides to the polymeric chain of RNA. If this were firmly proved many new possi- 

 bilities would be opened up, in particular the possibiUty of systematic experimental pro- 

 gress towards deciphering the peculiar code in which the structure of a protein is recorded 

 in the chain of a nucleic acid. As Chargaflf put it, we have no Rosetta stone for deciphering 

 this code. However, unlike the archaeologists, we can do experiments, asking questions 

 of Nature. Today, owing to the discoveries of Ochoa and Grunberg-Manago, we can 

 synthesize polynucleotides of simple structure. With their help it will be possible to 

 direct a systematic effort towards the elucidation of the 'chemical memory' of RNA, 

 based on experiment and not on general statistical considerations like those on which 

 Gamow tried, without much success, to base his work. 



