Specificity of tRNA-protein interactions in aminoacylation of tRNAs and translation initiation

Abstract

The interaction between nucleic acid and protein is crucial for the biological functions of cells. tRNAs provide an ideal system to study the specificity of such interactions. To expand our knowledge on the specific interaction between tRNA and aminoacyl-tRNA synthetase, the major recognition sites of E. coli tRNA{dollar}\sp{lcub}\rm Cys{rcub}{dollar} and tRNA{dollar}\sp{lcub}\rm Asn{rcub}{dollar} by their cognate synthetases were investigated. The anticodon, the discriminator base and the first base pair at the acceptor stem were demonstrated to be important for recognition of both tRNAs. Transplanting the anticodon (GCA) and the discriminator base (U73) of tRNA{dollar}\sp{lcub}\rm Cys{rcub}{dollar} to tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} converted tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} into a Cys acceptor, which initiated DHFR synthesis at a Cys initiation codon with cysteine. Transplanting the first base pair in the acceptor stem of tRNA{dollar}\sp{lcub}\rm Cys{rcub}{dollar}, G1:C72, to tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar}(GCA)/U73 greatly increased its aminoacylation level. Similarly, substitution of U35 with C35 or U36 with C36 abolished the aminoacylation of tRNA{dollar}\sp{lcub}\rm Asn{rcub}{dollar}; substitution of G34 with C34 converted tRNA{dollar}\sp{lcub}\rm Asn{rcub}{dollar} into a lysine acceptor. Replacement of G73 with A73, or U1:A72 with G1:C72 reduced the aminoacylation level; replacement of G73 with C73 or U73, or U1:A72 with U1xC72 destabilized the tRNA.;To understand the specific interaction of initiator tRNA with various protein components of the translation initiation machinery, a C1xA72 mismatch in the acceptor arm and three consecutive G:C base pairs in the anticodon stem, shown to be essential for initiation function of tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar}, were transplanted into the elongator tRNA{dollar}\sp{lcub}\rm Met{rcub}{dollar}(CUA) to generate Mi:2. Mi:2, though well aminoacylated, was not active in initiation of protein synthesis unless Methionyl-tRNA synthetase (MetRS) was also overproduced. It is demonstrated that Mi:2 is normally aminoacylated with lysine and that the failure of the Lysyl-tRNA to initiate protein synthesis is due to a block in formylation. Transfer of an additional base pair, G4:C69, and simultaneous overproduction of Methionyl-tRNA transformylase (MTF) and Lysyl-tRNA synthetase (LysRS) resulted in formylation of the Lysyl-tRNA and initiation of protein synthesis. This and additional data show that both the structural features and aminoacylation specificity contributes to formylation and initiation function of tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar}.