Transcription from the core promoter of the juvenile hormone esterase gene (-61 to +28) requires the presence of both an AT-rich motif (TATA box) and an initiator motif for any transcription to occur, when assayed by either transcription in vitro with lepidopteran Sf9 nuclear extracts or by transient-transfection assay in Sf9 cells. Additional gel-shift experiments indicated that at least one additional binding site is essential for transcription to occur. Mutational analysis in the transcription-in vitro and cell-transfection assays demonstrated that a 14-bp region from +13 to +27 relative to the transcription start site is also essential for transcription to occur. Whereas the wild-type core promoter is highly transcriptionally active, inclusion of additional flanking sequences to position -212 reduces that activity approx. 100-fold, and inclusion of the 5ʹ region out to position -500 reduces transcription by 200-fold. The pattern of dependence on both the AT-rich motif and the initiator for detectable transcription, and the high innate activity being repressed by 5ʹ-binding factors, was recapitulated in mosquito C7-10 cells. This study demonstrates that the cellular juvenile hormone esterase gene is organized as a composite core promoter, dependent on both TATA-box and initiator-binding factors, an organization that has been more commonly reported for viral promoters. This highly active composite core promoter is made more complex by the absolute dependence on the presence of a third site shortly downstream from the initiator, which is distinct from the ‘downstream promoter element’ described from some TATA-less genes. The juvenile hormone esterase gene thus appears to be a model of a cellular composite core promoter with a multipartite, indispensible requirement for not just both the TATA box and initiator, but also for at least a third core element as well.
Regulation of the juvenile hormone esterase gene by a composite core promoter
