The 3'-terminal end (NCCA) of tRNA determines the structure and stability of the aminoacyl acceptor stem.

Abstract

We have done a systematic study on the contribution of the single-stranded NCCA end (where N Is any nucleotide) to the stability of the aminoacyl stem of tRNA. A 7-bp RNA duplex with the single-strand ACCA 3' terminus derived from the aminoacyl stem of Escherichia coli tRNAA and several chemically synthesized sequence variants are characterized by proton NMR and thermodynamic parameters. The single-stranded 3' terminus noticeably stabilizes the duple in a sequence-dependent mer. Though the largest contribution to the stability gain due to the ACCA end is provided by the first dangling 3' nudeotide, the influence of even the fourth nucleotide is measurable. (7). One of these binding sites is localized in the acceptor stem; hence, we wanted to determine whether one could be detected in the acceptor-stem-derived microhelices. MATERIALS AND METHODSRNA oligonucleotides were synthesized chemically on a Gene Assembler Plus (Pharmacia) using H-phosphonate synthones according to a procedure described previously (8). The 5' and 2' OH groups of ribose were protected by 4,4'-dimethoxytrityl and tert-butyldimethylsilyl protecting groups, respectively. Protection of the exocycic amines in adenine and guanine was achieved by dimethylaminomethylene protection groups (8). The oligonucleotides were purified by HPLC on a Vydac C4 column. The RNA synthones were purchased from DIAGEN (Dusseldorf, F.R.G.). Singlestrand RNA concentrations for NMR measurements were typically 0.5-1.5 mM. The total volume of the samples was 0.5 ml. The buffer contained 100 mM NaCl and 10 mM sodium phosphate (pH 6.5). Where indicated, MgCl2 (5-7.5 mM) was added.The NMR spectra were determined on a Bruker AM 500 spectrometer operating at a proton resonance frequency of 500 MHz. To suppress the strong water signal, the 1-3-3-1 pulse sequence according to Hore (9) was applied. Chemical shifts are referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate.The UV absorbance measurements were carried out on a DU-8 spectrophotometer (Beckman) equipped with a variable-temperature unit. Absorbance at 260 nm was registered while heating 1-ml samples at a rate of 1 K/min. Single-strand RNA concentrations were 3 ,uM in all cases, and the buffer was the same as in the NMR samples. Thermodynamic parameters were determined on the basis of the two-state model from the temperature dependence of the equilibrium constant (10). RESULTS Chemical-Shift

Recognition by Tryptophanyl-tRNA Synthetases of Discriminator Base on tRNATrp from Three Biological Domains

“…This indicates the occurrence of a conformational change in the microenvironment of this base pair caused by the A73G mutation. This finding is consistent with the suggestions that N73 of tRNA can base-stack on the nearby first base pair of the tRNA acceptor stem helix and extend the stacking in this stem (22)(23)(24) and that N73 plays an important role in the stabilization of the acceptor stem helix by the single-stranded 3Ј terminus (25). Thus the conformational change in G1-C72 in bovine tRNA Trp elicited by the A73G mutation, as revealed by NMR, could be due to an effect on the base stacking between G73 and G1-C72.…”

Functional transfer RNAs with modifications in the 3'-CCA end: differential effects on aminoacylation and polypeptide synthesis.

“…However, the variant with a 3'-terminal guanosine, although fully chargeable, is a poor substrate for VRS. Some of the differences in aminoacylation activity of the 3'-end mutants may be due to effects of sequence variation at the 3'-CCA terminus on the structure and stability of the adjoining amino acid acceptor stem (25).…”

NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3' end.

“…The G1-C18 imino proton resonance of the U19 variant broadens at lower temperature (370C) than that of the A19 variant. This indicates a difference in the kinetics of imino proton exchange for the two variants (21). However, the stability of the terminal G1C18 base pair cannot be determined from the exchangeable proton kinetic behavior.…”