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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    if no figures are given, the bases are present in equimolar amounts. Poly-ABC, with no commas between the bases, has the sequence ABC repeated.) Since errors occur in cell free incorporation systems, a check is provided by the assignment of codons through an entirely different technique developed by Nirenberg and Leder in 1964. A trinucleotide of known sequence-derived either by degradation of RNA or prepared synthetically—causes specific molecules of aminoacyl-tRNA (transfer RNA carrying amino acids) to bind to ribosomes. Ribosomes are retained on filters of cellulose nitrate, but amino- acyl-tRNAs are not; the aminoacyl-tRNA species attached to ribosomes can therefore be separated from the unbound transfer molecules, which are removed by washing the filter with a salt solution. Each trinucleotide can be assayed by adding it to twenty different incubations, each containing ribosomes and one radioactive aminoacyl-tRNA species. This experiment has been performed for most of the triplets—an example is shown in figure 1.9—and the results are in fairly good agreement with the data obtained from polynucleotide directed amino acid incorporation experiments (summarized by Brimacombe et al., 1965; Nirenberg et al., 1966). The code which has been worked out from both sets of studies is shown in figure 1.10. But do these triplets code for the same amino acids in vivo ? Three types of experiment have suggested that most, and probably all, of these codons are used in E.coli with the meanings shown in the figure. Codon meanings can be deduced indirectly by analysing the amino acid changes produced in proteins by mutations. The tryptophan synthetase A protein of E.coli, for example, has been subjected to a detailed analysis of the amino acid substitutions which result from the mutation of a single nucleotide induced by a mutagen which causes a specific type of base change (see Berger, Brammar and Yanofsky, 1968). For example, if we know that tryptophan is coded by UGG and is mutated to arginine by inducing a U -» C base change, then CGG must be a cpdon used in vivo for arginine. A more productive way to use mutated proteins is to compare the amino acid sequences of wild type and frameshift mutants. Analysis of acridine induced double mutants in the lysozyme of phage T4 has proved very successful. When the sites of addition and deletion are fairly close to each other, the tryptic fingerprints of double mutant and wild type proteins differ only in those peptides derived from the frameshift region; these can be isolated and sequen ced with comparative ease. Using the codon assignments derived from studies in vitro, Streisinger et al. (1966) found that for every such double mutant a unique series of bases can be assigned to code for the wild type amino acid sequence and, with appropriate addition and deletion, for the double mutant amino acid sequence. Figure 1.11 shows an example ; a summary of the codons which have been shown to be used in vivo by phage T4 has been given by Ocada, Amagase and Tsugita (1970). The only direct way to assign codon meanings, of course, is to compare