A rapid method for mapping exposed cytosines in polyribonucleotides. Application to tRNATrp (yeast, beef liver)

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Abstract
A rapid method for mapping exposed cytosine residues in 5′-[32P]-labeled RNA molecules is suggested. The exposed cytosines (C's) are converted into uracyls (U's) by bisulphite treatment at pH 5.8 in the presence of Mg2+, followed by complete modification of the residual (non-exposed) C's by a methoxyamine and bisulphite mixture at pH 5.0. The control RNA is modified only by methoxyamine and bisulphite without the preliminary C→U conversion. The location of the exposed C's is determined by comparing the products of partial T1, T2, A and U2 ribonuclease digestions of the C → U converted and control RNAs after slab gel polyacrylamide electrophoresis and autoradiography. The method has been applied for mapping exposed cytosine bases in tRNATrp (yeast) which have been found in the anticodon loop and at the 3′-end of the molecule. In tRNATrp (beef liver), in addition to the same exposed bases, C in the diHU-loop is exposed. The data obtained are in full agreement with that is known about exposed C's for other tRNAs.