THE 3'-TERMINAL END (NCCA) OF TRANSFER-RNA DETERMINES THE STRUCTURE AND STABILITY OF THE AMINOACYL ACCEPTOR STEM

We have done a systematic study on the contribution of the single-stra nded NCCA end (where N is any nucleotide) to the stability of the amin oacyl stem of tRNA. A 7-bp RNA duplex with the single-strand ACCA 3' t erminus derived from the aminoacyl stem of Escherichia coli tRNA(Ala) and several chemically synthesized sequence variants are characterized by proton NMR and thermodynamic parameters. The single-stranded 3' te rminus noticeably stabilizes the duplex in a sequence-dependent manner . Though the largest contribution to the stability gain due to the ACC A end is provided by the first dangling 3' nucleotide, the influence o f even the fourth nucleotide is measurable. The nature of the N73 disc riminator base influences the stem structure and stability, which may be important for the recognition of tRNA by aminoacyl-tRNA synthetase. The stepwise attachment of the nucleotides to the 3' tail improves th e stacking of the unpaired bases over the helix stem. Hence, the ACCA end appears to be structured. Replacing Mg2+ with Mn2+ causes broadeni ng of certain imino proton peaks in the NMR spectrum, indicating a spe cific divalent metal ion binding site in the vicinity of the major ide ntity element of the duplex (G3-U70) that is required for its recognit ion by the Ala-tRNA synthetase.