Translation of R17 RNA by Escherichia coli ribosomes : Initiator transfer RNA-directed binding of 30 S subunits to the starting codon of the coat protein gene

Abstract

1. (1) Active ribosomes (tight vacant 50 S/30 S couples) not contaminated with subunits require factors IF-2 and IF-3 for initiation. 2. (2) Requirements for the formation of a 40 S initiation complex consisting of intact R17 RNA and fMet-tRNA bound to the 30 S subunit are fully satisfied by IF-3 alone, if 50 S subunits are absent. The 40 S complex, obtained in yields of up to 30% of the 30 S subunits added, is stable in sucrose gradients at 15 m m-Mg 2+ without fixation with glutaraldehyde. In the presence of 50 S subunits and GTP, the 40 S complex is converted quantitatively at 0 °C into a 76 S complex that is fully reactive with puromycin. 3. (3) In the presence of strongly associating 50 S-a subunits, IF-2 and GTP in addition to IF-3 are required for formation of the 40 S complex because IF-2 and GTP prevent reassociation of subunits by enhancing the binding of fMet-tRNA and IF-3 to the 30 S subunit in the absence of messenger. Optimal formation of the mRNA-free 30 S · fMet-tRNA complex, demonstrable on sucrose gradients even without fixation by glutaraldehyde, requires all three initiation factors and GTP. 4. (4) Binding of mRNA to 30 S subunits in the absence of initiator tRNA, observed on sucrose gradients after fixation, appears to be unspecific because it is not dependent on initiation factors. The results are compatible only with a model (Noll & Noll, 1972) in which the binding of initiator tRNA to the 30 S subunit directs the binding of the mRNA initiator codon. From these and earlier findings we visualise the sequence of assembly of an initiation complex between Escherichia coli ribosomes and R17 RNA as follows: (i) 70 S + IF-1 → 30 S · IF-1 + 50 S (Noll, 1972; Noll & Noll, 1972), (ii) 30 S · IF-1 + IF-3 → 30 S · IF-1,3, (iii) 30 S · IF-1,3 + fMet-tRNA · IF-2 · GTP → 30 S · IF-1,2,3 · GTP · fMet-tRNA, (iv) 30 S · IF-1,2,3 · GTP · fMet-tRNA + mRNA → 40 S 1 + IF-3, (v) 40 S 1 + 50 S → 76 S 1 + IF-1,2 + GDP.