FUNCTIONAL CIS-ELEMENT SEQUENCE REQUIREMENTS FOR SUPPRESSION OF GENE-EXPRESSION BY THE TNPA PROTEIN OF THE ZEA-MAYS TRANSPOSON EN SPM/

TNPA, one of the two transposition proteins encoded by the En/Spm tran sposable elements of Zea mays, suppresses the expression of genes that contain an appropriate cis element. Suppression can be monitored in t obacco protoplasts in a transient expression assay as follows. The pla nt promoter-driven expression of the Escherichia coli-glucuronidase (G US)-encoding gene, uidA, is repressed in the presence of TNPA if the G US gene contains a functional cis element in the untranslated RNA lead er sequence. Earlier, we found that the minimal cis element is compose d of two 12 bp sequences in a tail-to-tail inverted orientation. Each 12 bp sequence is sufficient to bind TNPA in vitro and can be thought of as a half-site in the cis element. Here, we investigated the sequen ce requirements of the minimal cis element. Our observations support o ur expectations that a functional cis element must provide a template to which two TNPA molecules can bind in the correct orientation. Seque nces within the half-sites can be altered as long as the eight bases t hat make up the consensus binding sites are not changed. However, we f ound the following unexpected sequence specificities. Firstly, some ch anges to the consensus binding sequence can be tolerated in one half-s ite, as long as the other site matches the consensus. Secondly, althou gh the region between the half-sites can vary in sequence and in lengt h between two and four bases, a thymidine residue is not tolerated dir ectly 5' preceding the second half-site. Since many variants of the ci s element sequence remain functional, the suppressor response element provides a flexible tool for artificially manipulating the expression of genes.