Question

Fakunding and Hershey mixed [³²P]IF2, [³H]fMet-tRNAfMet and AUG, an mRNA substitute, with 30S ribosomal subunits and either (a) GTP or (b) the unhydrolyzable GTP analog GDPCP. Then they centrifuged the mixtures in sucrose gradients and assayed each gradient fraction for radioactive IF2 (blue) and fMet-tRNAfMet (red). How to interpret the results that there is no difference for the binding of IF2 to the ribosome between the addition of GTP and GDPCP in the experiments?

 Figure 17.8 Formation of 30S initiation complex with GTP or GDPCP. Fakunding and Hershey mixed (2P]IF2, (H]f Met-tRNAMet and AUG, an mRNA substitute, with 30S ribosomal subunits and either (a) GTP or (b) the unhydrolyzable GTP analog GDPCP. Then they centrifuged the mixtures in sucrose gradients and assayed each gradient fraction for radioactive IF2 (blue) and fMet-TRNA Met (red). Both substances bound to 30S ribosomes equally well with GTP and GDPCP. (Source: Adapted from Fakunding, J.L. and J.W.B., Hershey, The interaction of radioactive initiation factor IF2 with ribosomes during initiation of protein synthesis. Journal of Biological Chemistry 248:4208, 1973.)

Answer 1

The interaction of initiation factor IF 2 with ribosomes during initiation of protein synthesis was studied in vitro with the radioactive factor, [ 32P]phosphoryl initiation factor IF 2. The binding and release of the factor were analyzed by sucrose density gradient centrifugation. The results show that: 1. Initiation factor IF 2 is part of the complete 30 S initiation complex formed in the presence of A-U-G, N formylmethionyl transfer RNA, initiation factor IF 1, and 5' guanosine triphosphate or 5' guanylyl methylenediphosphonate. 2. Initiation factor IF 2 binds to 30 S ribosomes in the absence of the other components of the 30 S initiation complex. The binding is stabilized by the addition of 5' guanosine triphosphate and by low salt concentrations in the gradients. 3. Initiation factors IF 1 and IF 3 greatly enhance the stability of initiation factor IF 2 binding to 30 S ribosomes. It is proposed that a complex of the 30 S ribosome and the three initiation factors may be an intermediate in the formation of the complete 30 S initiation complex. 4. Initiation factor IF 2 binds stoichiometrically to active 30 S ribosomes in the presence of saturating amounts of initiation factors IF 2, IF 1, and IF 3. If the binding reactions are treated with glutaraldehyde before gradient analysis, 2 molecules of initiation factor IF 2 are bound per 30 S ribosome. 5. Initiation factor IF2 and N formylmethionyl transfer RNA bind in equal proportions to the 70 S initiation complex formed with 5' guanylyl methylenediphosphonate. When the 70 S complex is formed with 5' guanosine triphosphate, however, initiation factor IF 2 no longer is bound to the complex. It is suggested that the role of 5' guanosine triphosphate hydrolysis is to increase the rate of ejection of initiation factor IF 2 from the 70 S initiation complex.