Enzymatic synthesis and some properties of a model primitive tRNA

Abstract

A model primitive tRNA with the nucleotide sequence GGCCAAAAAAAGGCCp was synthesized using T₄ RNA ligase. The nucleotide sequence of this newly synthesized oligonucleotide was confirmed by ladder analysis of several enzymatic digestion products. The secondary structure of the oligonucleotide was examined by comparison of the products of its digestion by single- and double-strand-specific nucleases with those of the digestion of the intermediate oligonucleotide GGCCAAAAAAA_OH. The results indicated that the two GGCC segments of the 5′ and 3′ ends of the model tRNA may form base pairs in solution. The same conclusion was derived from the result of affinitycolumn chromatography of the model oligonucleotide. When³²pGGCCAAAAAAAGGCC_OH was passed through a poly(U)-agarose column, about 70% of the applied sample bound to the poly(U)-agarose. In contrast, when the model oligonucleotide was passed through a poly(C)-agarose column, only 15% of the sample bound to the poly(C)-agarose. These results indicate that the newly synthesized oligonucleotide adopts a hairpin structure in solution. Two aspects of a potential biological activity of the synthetic model tRNA were examined. It was found that the oligonucleotide can bind to poly(U)-programmed 30S ribosomes and is recognized by Qβ replicase as a template for RNA synthesis.