Base pairing in wheat germ ribosomal 5S RNA as measured by ultraviolet absorption, circular dichroism, and Fourier-transform infrared spectrometry

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Abstract
UV absorption (UV) and circular dichroism (CD) spectra of wheat germ 5S RNA, when compared to tRNAPhe, indicate a largely base-paired and base-stacked helical structure, containing up to 36 base pairs. Fourier-transform IR (FT-IR) spectra of tRNAPhe and wheat germ ribosomal 5S RNA were acquired at 30.degree. and 90.degree. C. From the difference of the FT-IR spectra between 90.degree. and 30.degree. C, the number of base pairs in both RNA was determined by modification of a previously published procedure (Burkey, et al., 1983). The base-pair composition and total base-pair number from FT-IR data are not consistent for the 1st time with optical (UV, CD, Raman) and NMR results for ribosomal 5S RNA. Without added Mg2+, tRNAPhe gave 18 .+-. 2 base pairs [7 A-U and 11 G-C], in good agreement with the number of secondary base pairs from X-ray crystallography [8 A-U, 12 G-C and 1 G-U]. Within the 10% precision of the FT-IR method, wheat germ 5S RNA exhibits essentially the same number of base pairs [14 A-U, 17 G-C and 5 G-U; for a total of 36] in the absence of Mg2+ as in the presence of Mg2+ [14 A-U, 18 G-C and 3 G-U; for a total of 35], in agreement with the UV hyperchromism estimate of G-C/(A-U + G-C) = 0.58. Addition of Mg2+ increases the melting midpoint (Tm) for both A-U and G-C pairs in wheat germ 5S RNA. The average Tm for both types of base pairs increases from 48 (CD) or 54.degree. C (UV) in the absence of Mg2+ to 63 (CD) or 69.degree. C (UV) in the presence of Mg2+. From FT-IR, addition of Mg2+ increases the average base pair Tm by 11.degree. C for G-C pairs (56.degree. to 67.degree. C), but only by 4.degree. C for A-U pairs (54.degree. to 58.degree. C). The above results are consistent with 3 generalized eukaryotic 5S RNA secondary base-pairing schemes, of which the cloverleaf model gives the closest match.