Repression of IS200 transposase synthesis by RNA secondary structures

The IS200 transposase, a 16 kDa polypeptide encoded by the single open read ing frame (ORF) of the insertion element, has been identified using an expr ession system based on T7 RNA polymerase. In wild-type IS200, two sets of i nternal inverted repeats that generate RNA secondary structures provide two independent mechanisms for repression of transposase synthesis. The invert ed repeat located near the left end of IS200 is a transcriptional terminato r that terminates read-through transcripts before they reach the IS200 ORF. The terminator is functional in both directions and may terminate >80% of transcripts. Another control operates at the translational level: transposa se synthesis is inhibited by occlusion of the ribosome-binding site (RBS) o f the IS200 ORF, The RBS (5'-AGGGG-3') is occluded by formation of a mRNA s tem-loop structure whose 3' end is located only 3 nt upstream of the start codon, This mechanism reduces transposase synthesis similar to 10-fold. Pri mer extension experiments with AMV reverse transcriptase have provided evid ence that this stem-loop RNA structure is actually formed. Tight repression of transposase synthesis, achieved through synergistic mechanisms of negat ive control, may explain the unusually low transposition frequency of IS200 .