Phosphorothioate substitution–interference analyses. PhosphorImager printouts of selected and unselected tRNAs containing one of the four Rp-phosphorothioate nucleotides (thio-A, -C, -G, -U) separated on 10% polyacrylamide gels after iodine cleavage as indicated. The phosphorothioate-containing tRNAs were selected for P-site binding on 30S ribosomal subunits in the presence and absence of mRNA as indicated. Positions showing strong interference are indicated. T, unselected (total) phosphorothioate-containing tRNA population; S, selected (binding-competent) phosphorothioate-containing tRNA population. (A) tRNAPhe (E. coli). The sequence of the tRNA is shown in Fig. 2. (B) tRNAPhe from yeast. Only the bands corresponding to the anticodon region are presented, the sequence of the anticodon stem-loop region is 5′- CCAGACUGAAAAUCUGG-3′ (paired region are underlined, and anticodon in italics). (C) tRNAfMet (E. coli), anticodon region. The sequence of the anticodon stem-loop region is 5′-UCGGGCUCAUAACCCGA-3′. A specific mRNA containing a single AUG codon in the middle of a 46-nucleotide molecule (MF-mRNA) was used.