Regulation of the juvenile hormone esterase gene by a composite core promoter

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.

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Regulation of the juvenile hormone esterase gene by a composite core promoter

Biochemical Journal ◽

10.1042/0264-6021:3460233 ◽

2000 ◽

Vol 346 (1) ◽

pp. 233 ◽

Cited By ~ 6

Author(s):

Grace JONES ◽

Yan Xia CHU ◽

Douglas SCHELLING ◽

Davy JONES

Keyword(s):

Juvenile Hormone ◽

Core Promoter ◽

Juvenile Hormone Esterase ◽

Esterase Gene

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Functional Identification of the Transcription Start Site and the Core Promoter of the Juvenile Hormone Esterase Gene in Trichoplusia ni

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1995.2286 ◽

1995 ◽

Vol 214 (1) ◽

pp. 286-294 ◽

Cited By ~ 8

Author(s):

D. Schelling ◽

G. Jones

Keyword(s):

Juvenile Hormone ◽

Transcription Start Site ◽

Trichoplusia Ni ◽

Core Promoter ◽

Start Site ◽

Functional Identification ◽

Transcription Start ◽

Juvenile Hormone Esterase ◽

The Core ◽

Esterase Gene

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The Downstream Promoter Element DPE Appears To Be as Widely Used as the TATA Box in Drosophila Core Promoters

Molecular and Cellular Biology ◽

10.1128/mcb.20.13.4754-4764.2000 ◽

2000 ◽

Vol 20 (13) ◽

pp. 4754-4764 ◽

Cited By ~ 209

Author(s):

Alan K. Kutach ◽

James T. Kadonaga

Keyword(s):

Mutational Analysis ◽

Core Promoter ◽

In Vitro Transcription ◽

Tata Box ◽

Sequence Motif ◽

Promoter Element ◽

Core Promoters ◽

Downstream Promoter Element ◽

Biochemical Analyses

ABSTRACT The downstream promoter element (DPE) functions cooperatively with the initiator (Inr) for the binding of TFIID in the transcription of core promoters in the absence of a TATA box. We examined the properties of sequences that can function as a DPE as well as the range of promoters that use the DPE as a core promoter element. By using an in vitro transcription assay, we identified 17 new DPE-dependent promoters and found that all possessed identical spacing between the Inr and DPE. Moreover, mutational analysis indicated that the insertion or deletion of a single nucleotide between the Inr and DPE causes a reduction in transcriptional activity and TFIID binding. To explore the range of sequences that can function as a DPE, we constructed and analyzed randomized promoter libraries. These experiments yielded the DPE functional range set, which represents sequences that contribute to or are compatible with DPE function. We then analyzed the DPE functional range set in conjunction with a Drosophila core promoter database that we compiled from 205 promoters with accurately mapped start sites. Somewhat surprisingly, the DPE sequence motif is as common as the TATA box in Drosophila promoters. There is, in addition, a striking adherence of Inr sequences to the Inr consensus in DPE-containing promoters relative to DPE-less promoters. Furthermore, statistical and biochemical analyses indicated that a G nucleotide between the Inr and DPE contributes to transcription from DPE-containing promoters. Thus, these data reveal that the DPE exhibits a strict spacing requirement yet some sequence flexibility and appears to be as widely used as the TATA box in Drosophila.

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Transcription of the juvenile hormone esterase gene under the control of both an initiator and AT-rich motif

Biochemical Journal ◽

10.1042/bj3350079 ◽

1998 ◽

Vol 335 (1) ◽

pp. 79-84 ◽

Cited By ~ 16

Author(s):

Grace JONES ◽

Maria MANCZAK ◽

Douglas SCHELLING ◽

Helen TURNER ◽

Davy JONES

Keyword(s):

Core Promoter ◽

Tata Box ◽

Binding Motif ◽

Start Site ◽

Wild Type ◽

Transcription Start ◽

Juvenile Hormone Esterase ◽

A Cell ◽

Esterase Gene ◽

Wild Type Promoter

The binding of transcription factors to the core promoter of the juvenile hormone esterase gene was functionally characterized using both a cell-free in vitrotranscription functional assay and a cell transfection assay. A core JHE promoter (-61 to +28 bp relative to transcription start site) supported faithful transcription from the in vivo transcription start site. The nuclear extracts from the Sf9 insect cell line that provided transcription from that template also bound to that template as a probe in gel-mobility shift assays. Deletion or transversion of the initiator-binding motif (-1 to +4 bp) abolished detectable transcription either in vitro or in transfected cells. An AT-rich motif (ATATAT; -28 to -23 bp) serves another transcription factor-binding site. Mutation of the AT-rich motif to a canonical TATA-box preserved transcription, while either its deletion or complete transversion abolished or significantly reduced detectable transcriptional activity. These results indicate that, under these conditions, the functional operation of this core promoter approaches that of a composite promoter in which both the TATA- and initiator-binding protein complexes are necessary, even for basal transcription. On the other hand, these debilitating mutations to either the TATA box or initiator motif did not prevent the ability of the corresponding gel-shift competitive probes to compete with the wild-type promoter for binding by the transcription factors. Even a double transversion of both the AT-rich motif and the initiator-binding motif was able to competitively displace the protein complex that bound to the labelled wild-type probe. These data strongly indicate the presence of (an) additional core-promoter-associated transcription factor(s) (that is not the ‘downstream element ’) that contact(s) the AT-binding complex and/or initiator-binding factor with sufficient avidity to remove them from binding to the competing wild-type promoter sequence.

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