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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    sites on the ribosome. Peptidyl-tRNA must be bound at the P site before aminoacyl-tRNA can enter the A site. Bretscher (1966) and Bretscher and Marcker (1966) proposed that the initiator fmet-tRNA f possesses the ability to enter the P site directly; as figure 3.6 shows, the penultimate aminoacyl- rRNA then enters the A site and the first peptide bond is synthesised. There is no ambiguity since only fmet-tRNA f can respond to codons in the P site of 30S subunits and only met-tRNA m can respond to AUG codons in the A site of 70S ribosomes. Experiments with puromycin support this idea, since ribosome bound fmet-tRNA f is sensitive and reacts to give fmet-puro whereas met-tRNA m is comparatively insensitive under identical conditions. This suggests the fmet-tRNA f enters the P site and rnet-tRNA m enters the A site. That the blocked amino group of fmet-tRNA f plays some role in ensuring direct entry to the P site is suggested by the behaviour of peptidyl-tRNA analogues such as N-acetyl-phe-tRNA, which can mimic the initiator by entering the P site instead of the A site. The hybrid ribosome model discussed on page 53 and shown in figure 2.10 accounts for initiation on the supposition that the initiator binds to the 30p site. And as figure 2.22 shows, most of the P site seems in fact to be located on the 30S subunit. Addition of the 50S subunit to the initiation complex could then form a hybrid structure such as the 30p : 50a species of part (b) of figure 2.10. The hydrolysis of GTP then converts structure (b) into structure (d), that is converts the fmet-tRNA f into the reactive state shown by the puromycin reaction. Much data can be interpreted in terms of either the single entry or the double entry models, which are difficult to distinguish clearly by experi ments such as those with puromycin; but the double entry model in general provides a simpler explanation and is directly supported by more recent experiments which show there is no translocation at initiation. The behaviour of formyl-methionine when it joins the initiation complex suggests that the initiator enters the P site directly. Using the AUGGU 30 messenger described in the last chapter (page 68), Thach and Thach (1971) bound fmet-tRNA f to the ribosome-mRNA complex and then analysed the length of protected U n fragment to see whether a translocation is necessary to render the formyl-methionine reactive to puromycin. When GTP is used to bind the initiator tRNA to the ribosome, most (about 80%) of the formyl- methionine is released by puromycin; none reacts when GMP-PCP is used instead. But in each case, the same length of poly-U is protected; this is about 13 nucleotides, which indicates that the AUG codon is in the P site (see figure 2.17). Although the hydrolysis of GTP is therefore needed to activate the initiator, this reaction does not seem to involve a translocation: we may therefore conclude that fmet-tRNA f enters the P site directly. Similar results are obtained when fmet-tRNAf is bound to 30S subunits alone, which supports the idea that the major portion of the ribosome P site may be located on the 30S subunit.