Accessible and inaccessible bases in yeast phenylalanine transfer RNA as studied by chemical modification

Abstract

The selective modification of cytidine, uridine, guanosine and dihydrouridine residues in 32P-labelled yeast phenylalanine transfer RNA has been studied by the use of specific reagents. The selective modification of cytidine residues with the reagent methoxyamine is described. Of the six cytidines in the single-stranded regions of the cloverleaf formula, only two are completely reactive, C74 and C75 at the 3′-terminus. Cm32 in the anticodon loop is reactive to only a small extent. The selective modifications of uridine and guanosine residues with 1-cyclohexyl 3-[2-morpholino(4)-ethyl] carbodiimide methotosylate, is described. The reagent is also shown to be reactive with dihydrouridine. In the single-stranded regions of the secondary structure of yeast phenylalanine transfer RNA there are 16 base residues which this reagent could be specific for. However, only G20, Gm34 and U47 are extensively modified, whilst U33 and D16 are partially modified. G18 is modified to a very small extent. The results obtained in this study are also in good agreement with previous chemical modification studied by other workers, carried out on unlabelled yeast phenylalanine transfer RNA using different reagents to the ones described here. The pattern of chemical modification is compared with the three-dimensional structure obtained by an X-ray crystallographic analysis of the same tRNA species. The correlation between exposed regions of the model and the regions of chemical reactivity are everywhere consistent.