Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E-coli tmRNA

Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-transla tion reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 1 1 amino acid residue tag to the truncated protein that is a signal for degr adation and in recycling of the stalled ribosome. A tmRNA secondary structu re model with a partial tRNA-like structure and several pseudoknots was rec ently proposed. This report describes an extensive mutational analysis of o ne predicted pseudoknot (PK1) located upstream of the E. coil tmRNA tag-enc oded sequence. Both the extent of aminoacylation and the alanine incorporat ion into the tag sequence, reflecting the two functions of tmRNA, were meas ured in vitro for all the engineered RNA variants. To characterize structur e-function relationships for the tmRNA mutants, their solution conformation s were investigated by using structural probes and by measuring the tempera ture dependence of their UV absorbance. This analysis strongly supports the presence of a pseudoknot in E. coli tmRNA, and its involvement in trans-tr anslation. Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations. Mutations of the se cond stem of the pseudoknot also effect both functions. The nucleotide stre tch between the two stems (loop 2) is required for efficient trans-translat ion, and nucleotides at positions 61 and 62 must be guanine residues. The p robing data suggest the presence of magnesium ion(s) interacting with loop 2. The loops crossing the minor and major grooves can be mutated without si gnificant effects on tmRNA function. Nucleotide insertion or deletion betwe en the pseudoknot and the coding sequence do not change the mRNA frame of t he tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation. (C) 1999 Academic Press.