Nucleic Acids Research, 1990, Vol. 18, No. 10 2993
© 1990
MOLECULAR BIOLOGY |
Residual tRNA secondary structure in ‘denaturing‘ 8M urea/TBE polyacrylamide gels: effects on electrophoretic mobility and dependency on prior chemical modification of the tRNA
Department of Chemistry, Clark University 950 Main Street, Worcester, MA 01610, USA
Received December 4, 1989. Accepted February 20, 1990.
Fifteen individual species of tRNA were treated with the chemical modifiers diethylpyrocarbonate, 50%aqueous hydrazine or hydrazine/3 M NaCI. Following purification of the chemically modified material on polyacrylamide gels containing 8 M urea, variant minor bands, in addition to the expected main band, were observed for 12 of the 15 tRNAs. Characterization of the content of chemically altered bases In material recovered from such bands indicated that tRNAs containing modified nucleotides in base-paired stems were excluded from the main band and present, often in enhanced amounts, in the minor variant bands. The persistence of residual secondary structure on 8 M urea gels run at 45°C and the ability of chemically modified bases to alter electrophoretic mobilities warrant caution in designing and interpreting experiments in which chemically modified RNA is isolated on gels prior to further analysis. tRNAVal (VAC) was unique in that modified bases in non base-paired regions, according to the cloverieaf model of secondary structure, caused exclusion from the main band. Consequently, we propose a secondary structure for partially denatured tRNAVal(VAC), in which these bases are located indouble stranded regions of the molecule.