ABSTRACT
Binding of mRNA leader sequences to ribosomes was studied in conditions of a cell-free translation system based on wheat germ extract. Leader sequence of TMV mRNA (the so-called omega-RNA sequence) was able to bind simultaneously 80S ribosome and 40S ribosomal subunit. It was found that nucleotide substitutions in omega-RNA resulting in destabilization of RNA structure have no effect on the complex formation with both 80S ribosome and 40S ribosomal subunit. Leader sequence of globin mRNA is also able to form a similar joint complex. It is supposed that the ability of mRNA leader sequences to bind simultaneously 80S ribosome and 40S subunit is independent of leader nature and may reflect previously unknown eukaryotic mechanisms of translation initiation.
Subject(s)
5' Untranslated Regions , Eukaryota/genetics , Protein Biosynthesis , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Animals , Eukaryota/chemistry , Eukaryota/metabolism , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/geneticsABSTRACT
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.
