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Chemical and Enzymatic Probing of Spatial Structure of the Omega Leader of Tobacco Mosaic Virus RNA

N. E. Shirokikh, S. Ch. Agalarov, and A. S. Spirin*

Laboratory of Mechanisms of Protein Biosynthesis, Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; fax: (499) 632-7871; E-mail: spirin@vega.protres.ru

* To whom correspondence should be addressed.

Received November 12, 2009; Revision received December 3, 2009
The 5′-untranslated sequence of tobacco mosaic virus RNA – the so-called omega leader – exhibits features of a translational enhancer of homologous and heterologous mRNAs. The absence of guanylic residues, the presence of multiple trinucleotide CAA repeats in its central region, and the low predictable probability of the formation of an extensive secondary structure of the Watson–Crick type were reported as the peculiarities of the primary structure of the omega leader. In this work we performed chemical and enzymatic probing of the secondary structure of the omega leader. The isolated RNA comprising omega leader sequence was subjected to partial modifications with dimethyl sulfate and diethyl pyrocarbonate and partial hydrolyses with RNase A and RNase V1. The sites and the intensities of the modifications or the cleavages were detected and measured by the primer extension inhibition technique. The data obtained have demonstrated that RNase A, which attacks internucleotide bonds at the 3′ side of pyrimidine nucleotides, and diethyl pyrocarbonate, which modifies N7 of adenines not involved in stacking interactions, weakly affected the core region of omega leader sequence enriched with CAA-repeats, this directly indicating the existence of a stable spatial structure. The significant stability of the core region structure to RNase A and diethyl pyrocarbonate was accompanied by its complete resistance against RNase V1, which cleaves a polyribonucleotide chain involved in Watson–Crick double helices and generally all A-form RNA helices, thus being an evidence in favor of a non-Watson–Crick structure. The latter was confirmed by the full susceptibility of all adenines and cytosines of the omega polynucleotide chain to dimethyl sulfate, which exclusively modifies N1 of adenines and N3 of cytosines not involved in Watson–Crick interactions. Thus, our data have confirmed that (1) the regular (CAA)n sequence characteristic of the core region of the omega leader does form stable secondary structure, and (2) the structure formed is not the canonical double helix of the Watson–Crick type.
KEY WORDS: omega leader of TMV RNA, regular (САА)n polyribonucleotide, RNA triple helix, chemical modification of RNA, enzymatic cleavage of RNA, primer extension inhibition, diethyl pyrocarbonate, dimethyl sulfate, RNase A, RNase V1

DOI: 10.1134/S0006297910040024