Comparison of Escherichia coli tRNAPhe in the Free State, in the Ternary Complex and in the Ribosomal A and P Sites by Chemical Probing

Abstract

TRNA^Phe_E.coli was modified at accessible guanosine, cytidine, and adenosine residues using the chemical modification method described by Peattie and Gilbert [Proc. Natl Acad. Sci. USA, 77, 4679–4689 (1980)]. Modification characteristics of the tRNA in the free state, in the ternary complex with elongation factor EF‐Tu and GTP and in the ribosomal A and P sites were compared. A special procedure was devised to monitor, exclusively, tRNA molecules in the aminoacylated state. In the free tRNA, the most reactive bases are confined to the A₇₃‐C‐C‐A sequence of the aminoacyl stem, the anticodon loop, the D‐loop and the extra loop and the results correlate well with the three dimensional structure of tRNA^Phe_yeast determined by X‐ray studies. The pattern of reactivity was not affected either by charging the tRNA with phenylalanine or by labelling the 3′ terminus with pCp. In the ternary complex, with elongation factor EF‐Tu and GTP, changes in modification were observed at two sites, A₇₃‐C‐C‐A at the 3′ terminus and C‐13 and C‐17 in the D‐loop region, which are about 6 nm apart; no difference was observed in the anticodon loop. tRNA^Phe bound at the ribosomal A or P sites exhibited similar, but not identical, modification patterns. Whereas nucleotides C‐74 and C‐75 were strongly protected at both sites, the adjacent A‐73 showed an enhanced ractivity in the A site. The anticodon region G₃₄‐A‐A‐ms²i⁶ A was also strongly protected at both sites. In addition, nucleotide A‐21 was protected during A‐site, but not P‐site, binding.