CHARACTERIZATION OF IS1221 FROM MYCOPLASMA-HYORHINIS - EXPRESSION OF ITS PUTATIVE TRANSPOSASE IN ESCHERICHIA-COLI INCORPORATES A RIBOSOMAL FRAMESHIFT MECHANISM

Seven complete and two partial copies of IS 1221 variants from Mycopla sma hyorhinis and Mycoplasma hyopneumoniae characterized to date have established a consensus IS 1221 as a 1513 bp element with unique struc tural characteristics resembling the IS3 family of bacterial insertion sequences. Each IS 1221 copy contains highly conserved 28 bp imperfec t terminal inverted repeats and three distinctive internal inverted re peats (LIR, RIR and IIR). IIR is located within the coding region of t he element and it is proposed that it plays a critical role in the reg ulation of putative transposase expression. Consensus IS 1221 and one particular copy, G1135.2, contain a single long open reading frame (OR F). Two potential initiation codons are present at nucleotide 46 (AUG4 6) and nucleotide 397 (AUG397) and both are preceded by strong ribosom e-binding sites. Both initiation codons can be used efficiently in an Escherichia coli T7 expression system. The LIR has a negative regulato ry effect on translation initiation from AUG46. A -1 translational fra meshift event is shown to be involved in expression of the IS 1221 ORF and results in the production of 20 kDa and 6 kDa truncated proteins from the respective upstream initiation codons of the IS 1221 ORF. Bas e substitution and deletion mutations in sequences resembling characte rized motifs in documented examples of translational frameshifting res ulted in a significant increase in the full-length products and a corr esponding decrease in the truncated products from the IS 1221 ORF. In contrast to the usual -1 frameshift regulatory event in the IS3 family , which produces a transframe fusion product as the active transposase , IS 1221 may have evolved a high-frequency -1 frameshift mechanism th at produces a truncated product from the upstream coding domain and th ereby results in the regulated low-level production of the full-length presumptive transposase.