Heterogeneous nuclear ribonucleoprotein K is a transcription factor
Heterogeneous nuclear ribonucleoprotein K is a transcription factor
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DOI：
10.1128/mcb.16.5.2350
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发表时间：
1996-05-01
影响因子：
5.3
通讯作者：
Levens, D
中科院分区：
文献类型：
--
作者：
Michelotti, EF;Michelotti, GA;Levens, D
CT元件是c-myc基因上游的正性作用的同型嘧啶段，先前已经显示出与充分表征的转录因子Spl和异质核核糖核蛋白（hnRNP）K（一种与hnRNP复合物相关的不太充分表征的蛋白质）结合。本工作证明这两种分子都有助于CT元件激活的体外转录，CT元件的富含嘧啶的链既与hnRNP K结合又在体外竞争性抑制转录，表明hnRNP K通过该单链序列在激活转录中的作用。将重组hnRNP K直接添加到用具有单链CT元件的模板编程的反应混合物中增加了特异性RNA合成。如果hnRNP K是转录因子，则预测与RNA聚合酶II转录装置的相互作用。装有重组hnRNP K的亲和柱特异性结合转录激活所需的组分。耗尽的因素进行生化补充的粗TFIID磷酸纤维素馏分，表明hnRNP K可能与TATA结合蛋白（TBP）-TBP相关因子复合物相互作用。hnRNP K和表位标记的TBP之间在体内形成的复合物的免疫共沉淀以及重组蛋白之间在体外的结合证明了TBP和hnRNP K之间的蛋白质-蛋白质相互作用。此外，当这两种蛋白在体内过表达时，协同激活来自CT元件依赖性报告子的转录。这些数据表明，hnRNP K结合到一个特定的顺式元件，与RNA聚合酶II转录机制相互作用，并刺激转录，因此具有转录因子的所有特性。
The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind, The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro, The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence, Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted, Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K, Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.