Melting of Saccharomyces cerevisiae 5S ribonucleic acid: ultraviolet absorption, circular dichroism, and 360-MHz proton nuclear magnetic resonance spectroscopy

Abstract

The heat-induced melting of yeast 5S RNA and tRNAPhe has been monitored by UV, CD [circular dichroism] and 360 MHz 1H NMR spectroscopy to determine the extent of base stacking and base pairing in the native and denatured structures. In the presence of Mg2+, the optical data indicate .ltoreq. 40 base pairs in native yeast 5S RNA, a 60:40 ratio of GC to AU base pairs, with more single-stranded stacking and a slightly less stable structure (half-melted at 67.degree. C) than for tRNAPhe (half-melted at 71.degree. C). In the absence of Mg2+, the NMR results identify a minimum of .apprx. 32 base pairs at 25.degree. C (increasing to a minimum of .apprx. 35 base pairs in the presence of Mg2+), of which more than half are still intact at 48.degree. C. The native structure (25.degree. C) shows only minor dependence upon Mg2+ concentration, and no denatured forms could be detected. The present results support a previously proposed cloverleaf secondary structure for eukaryotic 5S RNA.