An NMR and mutational analysis of an RNA pseudoknot of Escherichia coli tmRNA involved in frans-translation

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Abstract
Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed frans-translation. According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350–400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes. A mutational analysis using a number of Escherichia colitmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in frans-translation. Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudo-knot structure in the presence of Mg2+. Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems. Structural instability of the pseudoknot structure, even in the presence of Mg2+, was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activities indicate that frans-translation requires the pseudoknot structure stabilized by Mg2+ and specific residues G61 and G62 in loop 3.