Experimental Evidence for the Genetic Code
In the 1960s, an American scientist, Marshall W.
Nirenberg, carried out ground breaking experimental
work that led to the discovery of the genetic code. The
code is represented as mRNA codons in the following
table:
If UGC is one of the codons for Cysteine, give all of the possible codons for serine:
UCU, UCC, UCA, UCG, AGU, AGC
Experiment 1
They created a cell-free extract by rupturing the cell walls of E.coli bacteria to
release their contents. DNA was destroyed using enzymes. The scientists produced
RNA comprised solely of uracil nucleotides (UUUUUUUUUU etc). They then added
this synthetic poly-U RNA into the cell-free extract. They set up 20 tubes each with
each of the 20 different amino acids in, but in each tube a different amino acid was
radioactively labelled. They incubated the tubes, and polypeptide synthesis occurred
in each. They then extracted the resulting polypeptides from the solutions.
One tube contained radioactive polypeptides. Which was the radioactive amino acid
that they had put into this tube? Explain you answer.
Amino acid: Phenylalanine
Explanation: The codon for Phenylalanine is UUU
Further experiments
Nirenberg assembled a team of scientists to crack the rest of the genetic code by
adding various synthetic RNAs to the cell free extract. For the following synthetic
RNAs which amino acids were incorporated into the resulting polypeptides?
Poly-A RNA incorporated: lysine
Poly-C RNA incorporated: proline.
Poly-U,C RNA (UCUCUCUCUCUC etc) incorporated: leucine and serine.
Further deductions
One main problem to work out was how many bases would be in codon. They knew
there was a total of four possible bases and 20 possible amino acids needed to be
coded for. How many amino acids could be coded for by the following number of
bases?
1 base – 4 amino acids
2 bases – 16 amino acids
3 bases – 64 amino acids
4 bases – 256 amino acids
Without any further experimental work, what could scientists deduce about codon
length?
Codons must be at least 3 bases long.
Later experiments
To determine a codon’s length, Niremberg created a synthetic cellulose filter that
ribosomes could attach to. tRNA, poly-U RNA and phenylalanine would wash
through. He poured 2 different mixtures over separate filters:
1. Ribosomes, tRNA and radioactive phenylalanine.
2. Ribosomes, tRNA, poly-U RNA and radioactive phenylalanine.
He poured through a washing solution and analysed the filters for radioactivity. Only
the filter from experiment 2 was radioactive. Explain why;
The ribosomes attach to poly-U RNA and tRNA, and start to synthesise radioactive
uracil. These complexes bind to the filter.
They then repeated this using poly-U RNA with 12 uracil-containing nucleotides
(UUUUUUUUUUUU) and found radioactivity.
They carried out further experiments using smaller and smaller chains. Chains of 6,
5, 4, 3 and 2 uracil-containing nucleotides resulted in radioactive in the filters. Which
polynucleotide length led to no radioactivity on the filter? Explain your answer.
Answer: 2 nucleotides.
Explanation: A codon must have 3 nucleotides to code for an amino acid.
More experiments
Next, they were able to synthesised further RNA with defined triplets of nucleotides.
They tested these with each of the 20 radioactive amino acids. Put ticks and
crosses on the table below to show which tubes led to the synthesis of radioactive
polypeptides.
trinucleotides
ACU
GCU
CCA
UCG
Alanine
Tubes containing these radioactive amino acids
Proline
Serine
Threonine
Final experiments
Once the code was solved, and while the sequencing was being finished, Nirenberg
turned to the examination of some questions about what it meant to have discovered
what people referred to as “the code of life.” One question that particularly fascinated
him was the issue of universality. He and his colleagues had done all their
experiments with bacteria. Experiments with plants, toads and guinea pigs showed
that the coding system was the same in each organism.
What is the significance of this finding?
Any organism that shares the same coding system probably shares a common
ancestor.
The table showing the genetic code can be used to construct mRNA for almost any
amino acid sequence. Construct a possible sequence for the following polypeptide:
polypeptide
mRNA
Met
Leu
Thr
Pro
Met
Gly
Ala