An estimate of the nearest neighbor base‐pair content of 5S RNA using CD and absorption spectroscopy

Abstract

We analyzed the CD and uv absorption spectra of 5S RNA from Escherichia coli using the method developed in the preceding paper. The analysis of spectra of 5S RNA at 20°C in 0.1 M NaClO₄, 2.5 mM Na⁺ (phosphate), pH 7.0, and 0.5 mM MgSO₄ gave 7 ± 3.6 A · U base pairs, 25 ± 3.6 G · C base pairs, and 7.5 ± 3.6 G · U base pairs. Estimates of nearest neighbor base pairs were more consistent with the Pieler–Erdmann and the Gewirth–Moore secondary structure models than with the Fox–Woese or the Burns–Luoma–Marshall models. We also examined the structure of 5S RNA as a function of temperature. The melting profile exhibited two transitions—one at about 35°C and one above 50°C. Our spectral data showed that helices I and II were stable during the first transition, and agreed with other data that helix III was the most likely helix to have melted. The results from this in-depth study of 5S RNA indicate that our method of analysis should be useful for studying the secondary structures of other small, unmodified RNAs.