Detection of a 16S rRNA . initiator-tRNA complex by a new selective labelling method

Cupric-ion induced hydrolysis of [35S]Met-tRNA but not of N-formyl-Met-tRNAMetf permitted the specific terminal labelling of initiator tRNA. Initiator tRNA, labeled in this way, was suitable for sequence analysis without the need for further purification. By probing labeled initiator tRNA with specific RNases, changes in this molecule during its interaction with the 30S particle or with 16S rRNA were investigated. Initiation complexes were resistant to the action of single-strand, base-specific nucleases Bc and Phy M and, except for one base of the anticodon stem, were also resistant to digestion by the double-strand-specific V1 nuclease of Naja venom. In contrast, T1 RNase digestion of the initiator tRNA in the presence of 16S rRNA enhanced cleavage of bases in the T stem of the molecule.