Initial Stages of the Thermal Unfolding of Yeast Phenylalanine Transfer RNA as Studied by Chemical Modification: The Effect of Magnesium

Abstract

The early stages of thermal unfolding of the tertiary structure of yeast tRNAPhe were followed, in the presence and absence of Mg2+, by measuring changes in the chemical accessibility of the bases uracil and guanine. The reagent used in these studies is 1-cyclohexyl 3-[2-morpholino(4)-ethyl]carbodiimide methotosylate. 32P-labeled tRNA was used so that the points of modification could be examined with RNase digestion and established fingerprinting techniques. Two regions of protection by Mg2+ were found. One is within the oligonucleotide U-A-m2G10 and the other is in the vicinity of residue U-59. The tertiary interactions and the D stem are the most readily melted parts of the tertiary structure. In the absence of Mg2+ the region of U-59 is the first part of the tertiary structure to become accessible to the reagent. This is closely followed by the opening up of the wobble G.cntdot.U base pair in the aminoacyl stem. Most of the triple interactions in the augmented D helix are also disrupted early in the melting. The region of intricate interactions between the invariant G-G part of the D loop and T-.PSI.-C-G loop contains the most stable set of tertiary structure interactions.