Dual roles of RNA helicase A in CREB-dependent transcription
Dual roles of RNA helicase A in CREB-dependent transcription
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DOI：
10.1128/mcb.21.14.4460-4469.2001
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发表时间：
2001-07-01
影响因子：
5.3
通讯作者：
Nakajima, T
中科院分区：
文献类型：
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作者：
Aratani, S;Fujii, R;Nakajima, T
RNA解旋酶A(RHA)是ATPase/DNA和RNA解旋酶家族的成员，是果蝇的同源物。无雄蛋白(MLE)，它调节X连锁基因的表达。RHA也是全息RNA聚合酶II(POI II)复合体的组成部分，将Pol II招募到CREB结合蛋白(CBP)。RH；I的ATPase和/或解旋酶活性是CREB依赖的转录所必需的。为了进一步了解RI-IA在基因表达中的作用，我们确定了一个由50个氨基酸组成的与POI II相互作用的反式激活结构域，并将其命名为最小反式激活结构域(MTAD)。该区域的蛋白质序列含有6个疏水残基，是RHA同源物所特有的，且保守。从全长RHA中删除该区域的突变体降低了CREB依赖转录的转录活性。此外，突变分析表明，MTAD中的几个色氨酸残基对于与Pol II的相互作用和反式激活是重要的，这些突变株具有与野生型RHA相当的ATP结合和ATPase活性。缺乏ATP结合活性的突变体仍能与Pol II相互作用。在依赖CREB的转录中，这些突变体的转录活性均低于野生型RHA，缺乏这两种功能的双重突变体的活性显著低于各自单独的突变体，并且双重突变体具有显性的负效应。这些结果表明，RHA可以通过Pol II的募集或通过ATP依赖的机制独立地调节(CREB依赖的转录)。
RNA helicase A (RHA) is a member of an ATPase/DNA and RNA helicase family and is a homologue of Drosophila. maleless protein (MLE), which regulates X-linked gene expression. RHA is also a component of holo-RNA polymerase II (PoI II) complexes and recruits Pol II to the CREB binding protein (CBP). The ATPase and/or helicase activity of RH;I is required for CREB-dependent transcription. To further understand the role of RI-IA on gene expression, we have identified a 50-amino-acid transactivation domain that interacts with PoI II and termed it the minimal transactivation domain (MTAD). The protein sequence of this region contains six hydrophobic residues and is unique to RHA homologues and well conserved. A mutant with this region deleted from full-length RHA decreased transcriptional activity in CREB-dependent transcription. In addition, mutational analyses revealed that several tryptophan residues in MTAD are important for the interaction with Pol II and transactivation, These mutants had ATP binding and ATPase activities comparable to those of wild-type RHA. A mutant lacking ATP binding activity mas still able to interact with Pol II. In CREB-dependent transcription, the transcriptional activity of each of these mutants was less than that of wild-type RHA, The activity of the double mutant lacking both functions was significantly lower than that of each mutant alone, and the double mutant had a dominant negative effect. These results suggest that RHA could independently regulate (CREB-dependent transcription either through recruitment of Pol II or by ATP-dependent mechanisms.