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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    prepares aminoacyl-tRNA for binding to the ribosome can therefore be represented as : Ts + Tu-GDP ^ Ts-Tu + GDP (figure 2.11) Ts-Tu + GTP ^ Tu-GTP + Ts (figure 2.12) aminoacyl-tRNA + Tu-GTP -» aminoacyl-tRNA-Tu-GTP (figure 2.13) When the GTP moiety of the aminoacyl-tRNA-Tu-GTP complex is cleaved at a later stage in protein synthesis (see below) the factor is released in the form of a Tu-GDP complex. Tu does not exist, therefore, in the form of a protein species free to combine as such with GDP or GTP; it oscillates between the two forms of binding GDP and GTP. The catalytic action of Ts in regenerating the Tu-GTP complex from the Tu-GDP complex achieves the cyclic use of Tu shown in figure 2.16. The interactions between Tu and GDP and Ts are reversible in vitro, but the reaction with aminoacyl-tRNA is irreversible and it is at the point of the third reaction above that the molecule is omitted to entering the cycle of protein synthesis. In the cell, of course, these reactions presumably proceed only in the direction of the cycle. Tu has been prepared in a homogeneous crystalline condition and has a molecular weight of42,000-44,000 daltons, this molecule binding one molecule of GDP. Ts has a molecular weight of 28,500 daltons and one cysteine which is necessary for catalytic activity. Using NEM (N-ethyl-maleimide) to inhibit -SH groups, Miller, Hachmann and Weissbach (1971) found that the ability of Tu to bind GDP can be destroyed, but that the complex of Tu-GDP is pro tected against this reaction. Ts also confers protection when added to Tu. This implies that Ts reacts with Tu at the same site where GDP is bound and utilises the same sulfhydryl group. The (different) sulfhydryl group needed to bind aminoacyl-tRNA is not protected in the Tu-GDP complex, which suggests that Tu has one binding site for GDP, GTP or Ts and another for aminoacyl- tRNA. Binding at the first site controls the activity of the second site. Binding Aminoacyl-tRNA to the Ribosome The complex which Tu-GTP (or S3-GTP) forms with aminoacyl-tRNA was shown by Lucas-Lenard and Haenni (1968) and Ono et al. (1969a) to transfer aminoacyl-tRNA to the ribosome; and by testing the reactivity to puromycin of phe-tRNA bound to ribosomes with poly-U, Weissbach, Redfield and Brot (1971a) were able to confirm that most, if not all, of the aminoacyl-tRNA is bound to the proper A site for entry. When the aminoacyl-tRNA-Tu-GTP moiety binds to ribosomes, the GTP is cleaved to yield phosphate and a Tu- GDP complex. The role of GTP in protein synthesis has been studied by substituting for it an analogue which cannot undergo hydrolysis; GMP-PCP (5' guanylyl- methylene-diphosphonate) has the structure shown in figure 2.14 in which a