Specific binding of Drosophila nuclear protein PEP (protein on ecdysone puffs) to hsp70 DNA and RNA.
The Drosophila protein PEP (protein on ecdysone puffs), a component hnRNP complexes, was previously immunocytologically localized on Drosophila giant chromosomes to puffs induced by ecdysone and to some heat shock-induced puffs (e.g. at the hsp70 locus at 87A7). Here, PEP was purified to homogeneity and characterized in its DNA and RNA binding features with specific reference to the hsp70 locus. In southwestern blotting assays, PEP was found to bind with high affinity to the hsp70 coding region, but not to a flanking region nor to the boundary elements scs and scs', and non-specifically to the intergenic hsp70 SAR. In UV cross-linking assays, PEP binds with even higher affinity to hsp70 transcripts, but not to transcripts of a flanking region or of a nearby gene, aurora . Finally, competition experiments indicate that PEP recognizes specific sequences within hsp70 mRNA; in these sequences two distinct motifs were found to be enriched. In summary, our results suggest the recognition of specific transcripts as a molecular basis for the association of the protein with specific hnRNP complexes.
Institut für Physiologische Chemie, Universitäts-Krankenhaus Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D006657:Histones / Q000096:biosynthesis; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009113:Muramidase / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000737:chemistry / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018840:HSP70 Heat-Shock Proteins; D006657:Histones; D019476:Insect Proteins; D009687:Nuclear Proteins; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C067400:PEP protein, Drosophila; D004247:DNA; D009113:Muramidase