Abstract
The requirement of initiation factors F1 (highly purified) and F2 (electrophoretically homogeneous) for ribosomal binding of N-formylmethionyl transfer RNA (fMet ∼ tRNA) at low Mg2+ concentration (3.5 mM), with the trinucleoside diphosphate ApUpG as messenger, was studied under various experimental conditions with 30S + 50S ribosomes and with 30S subunits alone. The results were qualitatively the same in both cases but the amount of binding was two to three times higher when both 30S and 50S subunits were present.
Although there was a virtually absolute requirement for F2 in all cases, considerable binding occurred at 0° in the absence of added F1. F1 addition stimulated binding up to twofold under these conditions. However, at 25°, the temperature at which the reaction is usually carried out, there was very little binding with F2 alone and addition of F1 stimulated the reaction five- to sixfold.
Contrary to current belief, the GTP analog 5′-guanylyldiphosphonate (GMP-PCP) cannot replace GTP in the binding reaction. In particular, there was but little stimulation of binding (about 1.5-fold) by addition of F1 to F2-containing samples when GMP-PCP was used. In marked contrast, binding was stimulated up to sevenfold by addition of F1 when GTP was substituted for the analog. Under these conditions, there was an ApUpG and F1-dependent hydrolysis of GTP. This is observable with 30S subunits alone and can hardly be related to the occurrence of translocation.
The results may be interpreted to mean that a complex relatively stable at 0°, but less stable at 25°, is formed upon addition of F2 alone. Conversion of the less stable to the more stable form of complex is made possible by addition of F1. This is accompanied or mediated by cleavage of GTP.
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Selected References
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