Co-occupancy of two Pumilio molecules on a single hunchback NRE.
Pumilio controls a number of processes in eukaryotes, including the translational repression of hunchback (hb) mRNA in early Drosophila embryos. The Pumilio Puf domain binds to a pair of 32 nucleotide (nt) Nanos response elements (NRE1 and NRE2) within the 3' untranslated region of hb mRNA. Despite the elucidation of structures of human Pumilio Puf domain in complex with hb RNA elements, the nature of hb mRNA recognition remains unclear. In particular, the site that mediates regulation in vivo is significantly larger than the 8-10-nt RNA elements bound to single Puf molecules in crystal structures. Here we present biophysical and biochemical data that partially resolve the paradox. We show that each NRE is composed of two binding sites (Box A and Box B) and that two Puf domains can co-occupy a single NRE. The Puf domains have a higher affinity for the 3' Box B site than the 5' Box A site; binding to the intact NRE appears to be cooperative (at least in some experiments). We suggest that the 2 Pumilio:1 NRE complex is the functional regulatory unit in vivo.
Gupta
Yogesh K
YK
Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Lee
Tammy H
TH
Edwards
Thomas A
TA
Escalante
Carlos R
CR
Kadyrova
Lyudmila Y
LY
Wharton
Robin P
RP
Aggarwal
Aneel K
AK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics*; D004330:Drosophila / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C471242:PUM1 protein, human; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D014157:Transcription Factors; C061245:hb protein, Drosophila; C074333:pum protein, Drosophila