Biochemical and biophysical analysis of pseudoknot-containing RNA fragments. Melting studies and NMR spectroscopy.

Three overlapping RNA fragments containing the pseudoknot, as found in the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA, have been isolated and purified. Site-directed cleavage of TYMV RNA by RNase H, followed by ammonium sulphate precipitation and ion-exchange HPLC, yielded a pure preparation of a 3'-terminal, 112-nucleotide TYMV RNA fragment. Transcription of TYMV cDNA by T7 RNA polymerase, resulted in the isolation of an 88-nucleotide fragment. Finally, a 44-nucleotide fragment containing the TYMV RNA pseudoknot and strongly resembling the aminoacyl acceptor arm of the viral RNA was also synthesised using T7 RNA polymerase. The three fragments were isolated in milligram amounts and used for biochemical structure mapping, ultraviolet melting studies and NMR spectroscopy. Chemical modification with diethyl pyrocarbonate and sodium bisulphite and enzymatic digestion with RNase T1 confirmed the presence of the pseudoknot in the 44-nucleotide fragment. Also the analogue of the T-stem and T-loop of the tRNA-like structure of TYMV RNA was found. The results of modification at various temperatures in Mg2+-containing buffers were in general agreement with optical melting studies. Ultraviolet melting analysis of the longer fragments revealed their greater complexity and the results appear similar to those obtained for some tRNA species. To obtain direct biophysical evidence for base-pairing and stacking interactions in the pseudoknot, NMR studies were initiated. The first proton-NMR spectra ever obtained for plant viral RNA fragments are presented. NMR spectra were recorded at various buffer conditions and at various temperatures. The spectra for the 112-nucleotide and 88-nucleotide fragment are too complicated to be solved at present. In the case of the 44-nucleotide fragment, however, the imino proton resonances are well separated and this system turns out to be most promising for structural studies.