Volume 1

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    extracts to achieve protein synthesis under direction of specific oligonucleotide templates in vitro. The earliest successful experiment was reported by Nirenberg and Matthaei in 1961; their system consisted of ribosomes and aminoacyl- tRNAs from E.coli, with ATP as energy source. Addition of synthetic polynucleotides to such systems stimulates the incorporation into polypeptides of specific amino acids; the effect of the template on the incorporation of any particular amino acid can be followed by using an incubation mixture in which this amino acid carries a C 14 radio active label but the other nineteen amino acids are unlabelled. Incorporation into protein is usually measured by precipitating the polypeptide synthesized and counting its radioactivity. Nirenberg and Mattaei found that the homo- polymer poly-U promotes the uptake of phenylalanine; this suggests that the codon for phenylalanine is UUU. Subsequent experiments have shown that poly-A encourages uptake of lysine and poly-C stimulates incorporation of proline (see Speyer, 1963). Poly-G is not easy to test because it is difficult to prepare a suitable physical form to use as template. The next advantage with such systems was the use of oligonucleotides con taining more than one type of base; such copolymers may direct the incorpora tion of several amino acids into protein. Polynucleotides of known overall composition but of random sequence are easily prepared by providing for the enzyme polynucleotide Phosphorylase a mixture of the appropriate nucleoside diphosphates; correlation between the frequencies of occurence of the various nucleotide triplets and the proportions of different amino acids in the poly peptide synthesized gives the nucleotide compositions of the codons. But the use of random copolymers suffers from the limitation that it shows only the nucleotide composition and not the sequence within the codon. A more powerful use of cell free systems has been derived from Khorana's synthesis of polynucleotides which consist of a known sequence of poly nucleotides. A copolymer containing two bases in alternating sequence causes the interdependent incorporation of only two amino acids when used as template. Jones, Nishimura and Khorana (1966) found that the kinetics of incorporation of the two amino acids are similar and approximately equimolar amounts of each are incorporated ; the incorporation of either amino acid is stimulated in the presence of the other and very much reduced by its absence. (The relatively low incorporation of one amino acid which takes place in the presence of the other is probably caused by some endogenous content in the extract system.) One such experiment was the stimulation of valine and cysteine incorporation by poly-UG shown in figure 1.8. If the nucleotide sequence UGUGUGUGUGUGUGUGUG directs synthesis of the polypeptide cys - val - cys - val - cys - val