Transcription of the juvenile hormone esterase gene under the control of both an initiator and AT-rich motif.
The binding of transcription factors to the core promoter of the juvenile hormone esterase gene was functionally characterized using both a cell-free in vitro transcription 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.
Jones
G
G
School of Biological Sciences, Molecular and Cellular Biology Section, University of Kentucky, Lexington, Kentucky 40506, USA. gjones@pop.uky.edu
Manczak
M
M
Schelling
D
D
Turner
H
H
Jones
D
D
D000818:Animals; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D018387:Codon, Initiator; D007605:Juvenile Hormones / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D011061:Poly A / Q000378:metabolism*; D011071:Poly T / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018411:Spodoptera; D013329:Structure-Activity Relationship; D016385:TATA Box; D014158:Transcription, Genetic
D018387:Codon, Initiator; D007605:Juvenile Hormones; D009841:Oligonucleotides; D011061:Poly A; D011071:Poly T; C008950:poly A-T; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase