Altered lead(II)-cleavage pattern of free Phe-tRNAPhe and Phe-tRNAPhe in ternary complex with EF-Tu:GTP

Pb2+ ions in sub-millimolar concentrations are known to cleave internucleotide bonds of phenylalanine-specific transfer RNA (tRNAPhe) from Saccharomyces. cerevisiae specifically between nucleotides D17 and G18 in the D-loop, with additional minor cleavages after D16 and G15. This makes lead(II) a sensitive structural probe for correct folding of tRNAPhe. In the present paper we use Pb2+ ions as a functional probe to determine whether this part of tRNA is protected by the Escherichia coli elongation factor EF-Tu in the ternary complex formed between Phe-tRNAPhe and EF-Tu.GTP. Our results show that for tRNA in complex with EF-Tu:GTP, the phosphodiester bond after D17 is cleaved, yet the phosphodiester bonds after D16 and G15 are not. To our knowledge, this is the first time that Pb2+ ions, bound at a specific site in tRNA, have been used both to investigate the correct folding of tRNA in complex, and to footprint a functional complex with components whose individual structures are known.