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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    activity of f2 is inhibited by reagents which interact with free sulfhydryl groups, but the addition of GTP or 30S subunits confers protection agains the inactiva- tion (Mazumder, Chae and Ochoa, 1969). This suggests that the factor has reactive -SH group(s) which become masked upon formation of a complex with GTP or when it binds to 30S subunits. The idea that its first action is to complex with GTP is supported also by the finding that pre-incubation of f2 with GTP before assay for factor activity results in a faster initial binding of fmet-tRNA f to ribosomes. Binding of fmet-tRNA f to the mRNA-30S subunit complex takes place in two stages, analogous to the interaction of aminoacyl-tRNA with 70S ribosomes. At 0°C, f2 promotes considerable binding of fmet-tRNA f to 30S subunits ; Chae, Mazumder and Ochoa ( 1969b) suggested that f2 and GTP may interact with fmet-tRNA f to form a transfer complex which then places the transfer species in the 30p site. This complex is not very stable; Lockwood, Chakraborty and Maitra (1971) found that it dissociates when attempts are made to isolate it on sephadex columns. Groner and Revel (1973) showed that f2 binds fmet-tRNA f by isolating a complex through centrifugation in glycerol gradients, but under less gentle conditions it is difficult to demonstrate complex formation directly. When f2 is used to bind fmet-tRNA f to ribosomes at 25°C, its activity is very much reduced; this accords with the idea that the transfer complex is not stable. However, Chae, Mazumder and Ochoa (1969b) found that the addition of f 1 increases binding very considerably. This suggests that the transfer complex may in some way be stabilised in the presence of fl. The role of f 1 is not clear, but Sabol et al. ( 1970) suggested that because it also appears to assist f3 to bind 30S subunits to mRNA its effect on fmet-tRNA f binding may be a secondary consequence of its direct action. That fl—the smallest initiation factor with a weight of only 9000 daltons—plays a secondary role in formation of the initiation complex is supported by experiments in which Hershey, Dewey and Thach (1969) showed that it binds to 30S subunits only as an integral part of the initiation complex. When fl is prepared from bacteria grown in a medium containing radioactive isotopes, the radioactive label associates with the ribosomes only when the reaction mixture contains all the components necessary for initiation. That the role of fl is confined to initiation is shown by its release from the initiation complex when 50S subunits are added to form the 70S ribosome. The function of fl may therefore be to stabilise the initiation complex as a whole rather than to participate directly in the reactions catalysed by f2 and f3. The role of GTP in the initiation complex has only recently been resolved. In early experiments the need for GTP seemed to depend upon the conditions of incubation, in particular upon the relative abundances of the different initiation factors, and some results suggested that GTP must be cleaved before fmet-tRNA f can bind to the 30S subunit. However, more recent work