Poly-U experiment

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In the poly-U experiment carried out by the US biochemist Marshall Nirenberg and his German post-doctoral student Heinrich Matthaei in 1961, a genetic coding unit was identified for the first time - the amino acid phenylalanine could be assigned to the base triplet UUU .

The decisive series of experiments in Nirenberg's laboratory at the NIH in Bethesda (Maryland) , which led to the identification of the first code on May 27, 1961 , was designed and carried out by Matthaei from May 15, 1961, so that, from a scientific perspective, the title “Father of genetic codes ”.

The experiment

In the so-called “triplet binding test” by Nirenberg and Matthaei, short pieces of messenger RNA (mRNA) with known base sequences were synthesized. The two researchers mixed these with ribosomes that had been isolated from bacteria . Investigations initially showed that ribosomes and mRNA attached to each other. Then Nirenberg and Matthaei fulfilled all the requirements for protein biosynthesis and put the necessary components in a test tube: Purified ribosomes, a mixture of all 20 amino acids (from experiment to experiment another amino acid was radioactively labeled) and finally a synthetic mRNA with one known base sequence. After all components had been put together and could act or react with one another, the mixture was placed on a filter that retained ribosomes, mRNA and newly synthesized protein, but allowed free amino acids through (filtrate). It was then examined whether there was radioactive material in the filtrate or whether it was being retained.

When an mRNA of the base sequence UUU was added to this cell-free system for gene expression and with radioactively labeled amino acid serine , no radioactivity was bound on the filter (i.e. no synthesis of poly-serine). However, if the radioactively labeled amino acid phenylalanine was added instead of serine in the next experiment , the radioactive signal was found on the filter (i.e. synthesis of poly-phenylalanine). In this way it was possible to prove that the base triplet UUU encodes the amino acid phenylalanine.

Using the same method, the amino acids lysine and proline could be assigned to the base triplet AAA and the triplet CCC . The codon GGG could not yet be deciphered in the experiment, due to its secondary structure , which meant that the base triplet could not be bound to the ribosomes.

In a further experiment, an mRNA with the base sequence UCU was added to the system. Radioactive serine was now found on the filter (i.e. synthesis of poly-serine) while radioactive phenylalanine was found in the filtrate (i.e. no synthesis of poly-phenylalanine).

From these results it could be concluded that when the base triplet matches the amino acid, protein biosynthesis takes place and the mRNA is translated .

Marshall Nirenberg received the Nobel Prize in Physiology or Medicine in 1968 together with Gobind Khorana and Robert Holley .

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proof

  1. ↑ Nirenberg, MW & Matthaei, JH (1961): The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. In: Proc. Natl. Acad. Sci. USA Vol. 47, pp. 1588-1602. PMID 14479932 ( doi : 10.1073 / pnas.47.10.1588 )
  2. ↑ See p. 231 (via Google Books ) in Hans-Jörg Rheinberger : Experimental systems and epistemic things. Wallstein Verlag, Göttingen 2001. ISBN 3-89244-454-4
  3. ↑ Abhinav Tiwari: 3 Kinds of Approaches by which the Genetic Code has been Cracked or Deciphered. In: Preserve article. Accessed April 14, 2019 .
  4. ↑ Information from the Nobel Foundation on the 1968 award to Marshall Nirenberg, Gobind Khorana and Robert Holley (English)