En/Spm encoded tnpA protein requires a specific target sequence for suppression.

The En/Spm encoded suppressor function has been reconstituted in transgenic tobacco protoplasts. The suppressor affects genes which contain an En/Spm responsive transposable element in the transcribed sequences. The En/Spm encoded protein tnpA binds a defined cis element in the inserted transposon, repressing expression of the adjacent gene. This was shown by monitoring transient expression of a bacterial marker gene (GUS) expressed from a strong plant viral promoter. Suppressible variants of the marker gene were produced by inserting I element sequences into the untranslated sequences of the GUS transcript. Comparison of transient expression of these variants in wildtype tobacco protoplasts with their expression in protoplasts transgenic for tnpA protein demonstrates that tnpA is the suppressor. In addition, the minimal cis element required for suppression has been defined as a dimer consisting of two 12 bp tnpA binding sequences in a particular inverted orientation. One of these dimers occurs in each En/Spm end close to the characteristic 13 bp terminal inverted repeat. TnpA binding sites in different arrangements do not respond as well to tnpA. The implications of this observation are discussed. This system can be used to analyse tnpA-DNA interactions involved in gene regulation further.