Direct differential absorbance profiles of denaturing transitions in ribosomal and mRNA

Abstract

SynopsisA direct method for measuring the derivative of thermal transition profiles (AAIAT), first described by Pavlov and Lyubchenko [Biopolymers 17, 795-798 (197811, is applied to the secondary structure of several eukaryotic ribosomal and messenger RNAs. The method consists of generating an effective AT between a sample and reference cuvette by altering the T, (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 a t different temperatures permits detailed examination of the base composition of differentially melting domains. We report here the AAlAT 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" polylrThe 18s and 28s rRNAs from chick embryos exhibit a reproducible series of peaks in the AAIAT profiles a t low salt with A T = 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 AAIAT profile in low salt. The stability of many of the transitions in each of the mRNAs is no greater than that of the secqndary 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.

The role of mRNA structure in the regulation of protein synthesis: Implications for studies of development

“…Therefore, a new explanation for differential initiation rates must be found [34]. The studies described here and elsewhere [54] support the notion that functional properties such as initiation, elongation, and termination rates are affected by the secondary and tertiary structural aspects of mRNA. It may be that modulation of gene expression can occur at the level of translation via the influence of mRNA structure on the rates of initiation, elongation, and termination.…”

“…The latter secondary structures are 43 and 46% base-paired for the CY and p globin mRNAs, respectively. These base-pairing values are significantly lower than have been estimated from optical melting profiles (58-60% for mixed a and p globin mRNAs [ 19,20,21]. The latter estimates were based on integrated melting profiles over the temperature range from 20°C to about 80°C.…”

“…Translation of mRNA can be affected by factors that affect RNA structure such as ionic conditions [ 181. Direct measurements by physical techniques [19][20][21][22], and enzymatic means [23,24] demonstrate that eukaryotic mRNA has considerable specific secondary structure. The experiments of Rosa [25] using enzymatic mapping techniques developed in this laboratory defined the secondary structures of initiation regions in three T7 phage late mRNA species, IIIb, IV and V. Although prokaryotic ribosomes locate the correct initiator through base-pairing between the 3'-end of 16s rRNA and the region immediately preceding the AUG, the factors that affect the translational efficiency of an mRNA are unclear.…”