Regulatory elements in the juvenile hormone binding protein gene from Galleria mellonella--topography of binding sites for Usp and EcRDBD.
The juvenile hormone binding protein (JHBP) plays a key role in the protection and transport of the hormone to target tissues. In this report the sequence of the jhbp promoter comprising about 2000 bp is characterized. Using a minimized false positive algorithm, six putative regulatory elements, Hunchback, Heat shock factor binding element, Ultrabithorax, Broad-Complex Z3, Elf-1 and Chorion factor 1/ultraspiracle (CF1/Usp) were found in the distal promoter of the jhbp gene. Proteins from nuclear extract of Galleria mellonella fat body form four specific complexes with probe containing TATA box, five complexes with Inr probe and one protein complex with DPE probe. EMSA and footprinting analyses showed that one of the three CF1/Usp elements (starting at -1053) has an exceptionally high affinity to Usp protein. An unknown, high-affinity Usp/EcRDBD-binding element (TCAACA-AAC-TGTTCA), distinct from 20-hydroxyecdysone response elements, was identified in the jhbp gene promoter, based on a footprinting assay. Deletions of jhbp promoter in the regions containing the CF1/Usp elements enhance the transcriptional activity of luciferase reporter gene in the Trichoplusia ni High Five cell line. Obtained data suggest that jhbp promoter is TATA- and Inr-driven, CF1/Usp elements exhibit inhibitory effect on jhbp expression, and an interaction between Usp and DNA relies on recognition of the consensus sequence (GGGTCA) and on ionic interactions of several phosphate groups outside from this element.
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspiańskiego 27, 50-370 Wrocław, Poland.
Agnieszka J
AJ
Sok
Grazyna
G
Andruszewska
Anna
A
Niewiadomska-Cimicka
Iwona
I
Grad
Grzegorz
G
Rymarczyk
Dorota
D
Pajdzik
Marek
M
Orłowski
Marcin T
MT
Schmidt
Włodzimierz
W
Grajek
Andrzej
A
Ozyhar
Marian
M
Kochman
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D017344:Genes, Insect; D019476:Insect Proteins / Q000235:genetics*; D007605:Juvenile Hormones / Q000378:metabolism*; D018402:Manduca / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics* / Q000378:metabolism*; D046912:Multiprotein Complexes; D011401:Promoter Regions, Genetic; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D050436:Regulatory Elements, Transcriptional; D013045:Species Specificity; D016385:TATA Box; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; D007605:Juvenile Hormones; D046912:Multiprotein Complexes; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor; C035606:juvenile hormone-binding protein, insect; D004247:DNA