Probing a tRNA core that contributes to aminoacylation

The contribution of the tRNA "core" to aminoacylation is beginning to be re cognized. One example is the core region of Escherichia coli tRNA(Cys) whic h has been shown by biochemical studies to be important to; aminoacylation. This core has several layers of unusual base-pairs, which are revealed by the recent crystal structure of the tRNA complexed with the elongation fact or EF-Tu and an analog of GTP. One of these layers consists of a 9:[13:22] base-triple, rather than the 46:[13:22] or 45:[13:22] base-triple that is c ommonly observed in tRNA structure. Because 13:22 is an important element i n aminoacylation of E. coli tRNA(Cys), a better understanding of its struct ure in the tRNA core will shed light on its role in aminoacylation. In this study, we used the phage T7 transcript of the tRNA as a substrate. We prob ed the structure of 13:22 by dimethyl sulfate and tested its partner in a b ase-triple by generating mutations that could be assayed for aminoacylation . The results of this study in general are in a better agreement with a 46: [13:22] base-triple that we previously proposed. Although these results are not interpreted as direct proof for the 46:[13:22] base-triple, they shed new light on features of the tRNA core that are important for aminoacylatio n. (C) 2000 Academic Press.