Hamann S, Strätling W H
Institut für Physiologische Chemie, Universitäts-Krankenhaus Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Nucleic Acids Res. 1998 Sep 15;26(18):4108-15. doi: 10.1093/nar/26.18.4108.
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.
果蝇蛋白PEP（蜕皮激素胀泡上的蛋白）是hnRNP复合物的一个组成部分，此前通过免疫细胞化学方法定位在果蝇巨型染色体上由蜕皮激素诱导产生的胀泡以及一些热休克诱导产生的胀泡上（例如位于87A7的hsp70基因座处）。在此，PEP被纯化至同质，并针对hsp70基因座对其DNA和RNA结合特性进行了表征。在蛋白质印迹法分析中，发现PEP与hsp70编码区具有高亲和力结合，但与侧翼区以及边界元件scs和scs'不结合，且与基因间hsp70 SAR非特异性结合。在紫外线交联分析中，PEP与hsp70转录本的结合亲和力更高，但与侧翼区或附近基因极光（aurora）的转录本不结合。最后，竞争实验表明PEP识别hsp70 mRNA内的特定序列；在这些序列中发现两个不同的基序富集。总之，我们的结果表明对特定转录本的识别是该蛋白与特定hnRNP复合物结合的分子基础。
