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

Transfer-messenger RNA (tmRNA) is a unique molecule that combines propertie s from both tRNA and mRNA, and facilitates a novel translation reaction ter med trans-translation. According to phylogenetic sequence analysis among va rious bacteria and chemical probing analysis, the secondary structure of th e 350-400 nt RNA is commonly characterized by a tRNA-like structure, and fo ur pseudoknots with different sizes. A mutational analysis using a number o f Escherichia coli tmRNA variants as well as a chemical probing analysis ha s recently demonstrated not only the presence of the smallest pseudoknot, P K1, upstream of the internal coding region, but also its direct implication in trans-translation. Here, NMR methods were used to investigate the struc ture of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide sub stitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudoknot struc ture in the presence of Mg2+. Imino proton resonances were observed consist ent with formation of two helical stem regions and these stems stacked to e ach other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleotides, loop 3, between the stems. Structural in stability of the pseudoknot structure, even in the presence of Mg2+, was fo und in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activi ties indicate that trans-translation requires the pseudoknot structure stab ilized by Mg2+ and specific residues G61 and G62 in loop 3.