Relationship between size of mRNA ribosomal binding site and initiation factor function

The rate and the extent of the binding of initiator fMet-tRNA(fMet) to 30S ribosomal subunits in the presence of IF1, IF2 and GTP is either inhibited or slightly stimulated by the presence of IF3 depending on whether the initiation triplet AUG or the polynucleotide poly(AUG) is used as template. To determine the length of the template required for the transition from the AUG- to the poly(AUG)-type of behavior in the presence of IF3, the ribosomal binding of fMet-tRNA was studied in response to AUG triplets extended on either the 5'- or the 3'-side by stretches of homo-oligonucleotides of different lengths. When the binding of fMet-tRNA was studied at equilibrium it was found that IF3 no longer inhibits the amount of ternary complex formed if AUG is extended either 10 nucleotides on the 5'- or 35-40 nucleotides on the 3'-side. When the initial rate of ternary complex formation is considered, shorter extensions (4 nucleotides on the 5'-side or 20-30 nucleotides on the 3'-side) are sufficient to elicit a substantial stimulation by IF3. These results are discussed in relation to the mechanism of action of the initiation factors in the selection of the initiation region of the mRNA by ribosomes.