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 have been 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‐labelled tRNA was used so that the points of modification could be examined with ribonuclease digestion and established fingerprinting techniques. Two regions of protection by Mg2+ have been found. One is within the oligonucleotide U8‐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 · 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 the T‐Ψ‐C‐G loop contains the most stable set of tertiary structure interactions. Copyright © 1977, Wiley Blackwell. All rights reserved