Transcriptional activation of the rice tungro bacilliform virus gene is critically dependent on an activator element located immediately upstream of the TATA box

To investigate the transcriptional mechanisms of rice tungro bacilliform vi rus, we have systematically analyzed an activator element located immediate ly upstream of the TATA box in the rice tungro bacilliform virus promoter a nd its cognate trans-acting factors. Using electrophoretic mobility shift a ssays, we showed that rice nuclear proteins bind to the activator element, forming multiple specific DNA-protein complexes via protein-protein interac tions, Copper-phenanthroline footprinting and DNA methylation interference analysis indicated that multiple DNA-protein complexes share a common bindi ng site located between positions -60 to -39, and the proteins contact the activator element in the major groove. DNA UV cross-linking assays further showed that two nuclear proteins (36 and 33 kDa), found in rice cell suspen sion and shoot nuclear extracts, and one (27 kDa), present in root nuclear extracts, bind to this activator element. In protoplasts derived from a ric e (Oryza sativa) suspension culture, the activator element is a prerequisit e for promoter activity and its function is critically dependent on its pos ition relative to the TATA box. Thus, transcriptional activation may functi on via interactions with the basal transcriptional machinery, and we propos e that this activation is mediated by protein-protein interactions in a pos ition-dependent mechanism.