You perform a cell free translation experiment like Nirenber

You perform a cell free translation experiment like Nirenberg and Matthaei, but you forget to write down what nucleotides you added to make the mRNA. You precipitate the translated polypeptides and measure the relative amount of radiolabeled amino acids incorporated into them. You get 25% proline, 25% threonine, 12.5% glutamine, 12.5% lysine, 12.5% asparagine, and 12.5% histidine. What nucleotides and in what % did you add to make the mRNA? Please explain step by step.

Why is the answer 50% C and 50% A?

Solution

The codon that codes for the amino acids are as follows:

a) Proline: CCU, CCA. CCG, CCC

b) Threonine: ACU, ACG, ACC, ACA

c) Glutamine: CAA, CAG

d) Lysine: AAA, AAG

e) Asparagine: AAU, AAC

f) Histidine: CAU,CAC

In a cell free extraction system used by Nirenberg and Matthaei, E.coli extract (with DNA removed) is added to 20 tubes. All amino acids attached to tRNA are added to each tube, except one amino acid is radioactively labeled. Synthetic RNA consisting of either single or combination of nucleotides is added. F or example, poly U, poly A, poly G, Poly C, Poly AG, poly AC, Poly AU, Poly AGC etc. The radioactivity was counted for each tube.

If the synthetic RNA is made up of 50% A and 50% C, then the possible triplet codons will be,

AAA, AAC, ACA, ACC, CCC, CCA, CAA, CAC.They code for:

AAA: Lysine

AAC: Asparagine

ACC: Threonine

ACA: Threonine

CCC: Proline

CCA: Proline

CAC: Histidine

CAA: Glutamine

All these amino acids give the expected percentages that were obtained in the mixture. Threonine and Proline are two times in amount as they are coded by two codons. Hence, their amounts are 25%. The other four amino acids, asparagine, glutamine, lysine and Histidine are coded by one codon. Hence, they are obtained at 12.5% amounts. Total is 100 %.

Hence, it is 50% A and 50% C.