Direct differential absorbance profiles of denaturing transitions in ribosomal and mRNA.

Abstract

AbstractA direct method for measuring the derivative of thermal transition profiles (ΔA/ΔT), first described by Pavlov and Lyubchenko [Biopolymers 17, 795–798 (1978)], is applied to the secondary structure of several eukaryotic ribosomal and messenger RNAs. The method consists of generating an effective ΔT between a sample and reference cuvette by altering the Tm (midpoint denaturation temperature) of one of the solutions with respect to the other. This can be done by changing salt concentration, solvent, pH, or ligands. Scanning the two cuvettes by varying the wavelength at different temperatures permits detailed examination of the base composition of differentially melting domains. We report here the ΔA/ΔT profiles generated by monovalent ion concentration differences for a number of high‐molecular‐weight natural RNAs, as well as the synthetic polynucleotides poly(rA) and “random” poly[r(A,G,U,C)].The 18S and 28S rRNAs from chick embryos exhibit a reproducible series of peaks in the ΔA/ΔT profiles at low salt with ΔT = 4K. The high‐temperature transitions in 28S rRNA appear to contain G·C base pairs exclusively, in contrast to those in 18S rRNA or any natural mRNA. Each mRNA we have examined (bacteriophage MS2, globin mRNA from rabbit reticulocytes, and procollagen mRNA from chick embryos) exhibits a distinctive ΔA/ΔT profile in low salt. The stability of many of the transitions in each of the mRNAs is no greater than that of the secondary structure in random poly(A,G,U,C) in low salt. More than 50% of the base pairing in procollagen mRNA actually “melts” below the mean for the random copolymer, indicating that despite its high G·C content, this mRNA contains a secondary structure that is exceptionally low in stability.