Flexibility of end-labeled polymers from electron spin resonance line-shape analysis: 3' terminus of transfer ribonucleic acid and 5S ribonucleic acid

Abstract

Saccharomyces cerevisiae tRNA and 5S RNA, Escherichia coli 5S RNA, and wheat germ 5S RNA have each been specifically spin-labeled at the 3'-terminal ribose to give morpholino-spin-labeled (MSL) RNAs. Enzymatic hydrolysis with pancreatic RNase, followed by anion-exchange chromatography, confirms the site of attachment of the spin-label. Effective rotational correlation times, TB and TC, have been determined from electron spin resonance (ESR) peak heights and widths as a function of temperature for each MSL RNA, and Arrhenius plots of -log T vs. 1/T have been constructed. TC is a measure of internal flexibility at the link between the label and the RNA, while TB is a measure of rotational flexibility of the RNA near the labeled site. Validity of the TB and TC determination has been confirmed from simulation of the experimental EPR spectra by theoretical spectra computed for various attachment geometries and motional rates. Discontinuities in the slope of Arrhenius plots for TB were seen at 34 and 66 degrees C (yeast MSL tRNA), 37 and 60 degrees C (E. coli MSL 5S RNA), 37 and 57 degrees C (yeast MSL 5S RNA), and 36 and 54 degrees C (wheat germ MSL 5S RNA). Temperature-induced hydrolysis of each MSL RNA was less than 5% as determined by gel-filtration chromatography. The melting curves are consistent with a recently proposed universal secondary structural model for prokaryotic and eukaryotic 5S RNA.