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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    depend on the amount of f2 present. When both 30S and 50S subunits are present and low catalytic amounts of f2 are used, GMP-PCP cannot replace GTP and its substitution does not allow binding of fmet-tRNA f . But at higher levels of f2 when the factor acts stoichiometrically, GMP-PCP can substitute for GTP with about 50% efficiency. Although fmet-tRNA f then sediments at the 70S position in association with ribosomes, the formyl-methionine moiety is not available to puromycin. When the GMP-PCP is removed by isolating the complex under conditions which cause loss of GTP, the fmet-tRNA f again becomes reactive to puromycin. Retention of the nucleotide by the initiation complex may therefore prevent fmet-tRNAf from reacting with puromycin ; GTP hydrolysis may be needed to release factor f2 from the ribosome. When release cannot take place because the GMP-PCP cannot be hydrolysed, the ribosome bound fmet-tRNA f is unreactive and the f2 protein cannot promote binding of initiator to any further complexes. This reduces the overall binding to a very small level when only catalytic amounts of f2 are present and ensures that the fmet-tRNA f which is bound cannot interact with puromycin. When larger amounts of f2 are present, of course, each factor can bind fmet-tRNA f to the initiation com plex, although the formyl-methionine is unreactive because of the failure of the complex to release its GMP-PCP and f2. This model has been supported by experiments in which the location of f2 has been examined after initiation. Lockwood, Sarkar and Maitra (1972) found that when GTP is used to promote binding of fmet-tRNA f to 30S subunits, the f2 factor is released from the 70S ribosomes as soon as 50S subunits are added. But when GMP-PCP is substituted, the f2 factor remains attached to the 70S ribosomes. This supports the idea that release of initiation factors is associated with a change in the conformation of the ribosome and that both events rely upon the cleavage of GTP. By following the release of labelled f2 from ribosomes after initiation, Fakunding and Hershey (1973) have suggested that the role of GTP hydrolysis is to increase its rate of removal from the 70S particle. Ribosome Binding to the Messenger Initiation factor f3 is needed to bind 30S subunits to messenger RNA; the effect of f3, which has a molecular weight of 21,200 daltons and one essential -SH group, is enhanced when f 1 is present. One role of this factor is therefore to help 30S subunits to recognise initiation sites and to prevent them from binding securely to other sequences containing AUG codons. Dubnoff, Lockwood and Maitra (1972) have suggested that f3 does not prevent the formation as such of incorrect complexes, but destabilizes the binding of 30S subunits at false locations so that they leave the messenger and are free to seek another binding site. The first observation which implied that there may be more than one class