Interaction of Individual Structural Domains of hnRNP LL with the BCL2 Promoter i-Motif DNA
Interaction of Individual Structural Domains of hnRNP LL with the BCL2 Promoter i-Motif DNA
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DOI：
10.1021/jacs.6b05036
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发表时间：
2016-08-31
影响因子：
15
通讯作者：
Hecht, Sidney M.
中科院分区：
文献类型：
--
作者：
Roy, Basab;Talukder, Poulami;Hecht, Sidney M.
最近发现BCL 2（B细胞淋巴瘤2基因）启动子i基序DNA通过与核糖核蛋白hnRNP L样（hnRNP LL）的相互作用在基因表达调节中的作用，促使对这种蛋白质DNA相互作用的性质进行更详细的研究。hnRNP LL的RNA识别基序（RRM）单独表达，并且发现RRM 1和RRM 2都有效地结合到BCL 2 i基序DNA，并且对于转录激活至关重要，而RRM 3 -4仅与该DNA弱结合。结合后，通过CD光谱的变化监测DNA的解折叠。i-基序DNA的突变分析显示，结合主要涉及的i-基序的侧环。通过在蛋白质和DNA混合后的预定时间记录CD光谱来探索DNA RRM 1结合的动力学。摩尔椭圆率的变化在30 s后很明显，并在1 min内基本完成。通过在RRM 1的137位引入荧光供体6-CNTrp，并在i-基序DNA中引入受体4-amingbenzo[g]quinazoline-2-one（C-f）代替胞苷（22），获得了蛋白质DNA相互作用的更详细视图。通过FRET监测两种物质的结合过程，其反映了在几分钟的时间内能量转移的稳定增加。FRET信号可以通过进一步添加（未标记的）RRM 2而减弱，这无疑反映了与i基序DNA结合的竞争。这些使用来自hnRNP LL的各个RRM结构域的实验证实了该转录因子在通过启动子元件中的i基序激活BCL 2转录中的作用。
The recently discovered role of the BCL2 (B-cell lymphoma 2 gene) promoter i-motif DNA in modulation of:gene expression Via interaction with the ribonucleoprotein hnRNP L-like (hnRNP LL) has prompted a more detailed study of the nature of this protein DNA interaction. The RNA recognition motifs (RRMs) of hnRNP LL were expressed individually, and both RRM1 and RRM2 were found to bind efficiently to the BCL2 i-motif DNA, as well as being critical for transcriptional activation, whereas RRM3-4 bound only weakly to this DNA. Binding was followed by unfolding of the DNA as monitored by changes in the CD spectrum. Mutational analysis of the i-motif DNA revealed that binding involved primarily the lateral loops of the i-motif. The kinetics of binding of the DNA RRM1 was explored by recording CD spectra at predetermined times following admixture of the protein and DNA. The Change in molar ellipticity was readily apparent after 30 s and largely complete within 1 min. A more detailed view of protein DNA interaction was obtained by introducing the fluorescence donor 6-CNTrp in RRM1 at position 137, and the acceptor 4-amingbenzo[g]quinazoline-2-one (C-f) in lieu of cytidine(22) in the i-motif DNA. The course of binding of the two species was monitored by FRET, which reflected a steady increase in energy transfer over a period of several minutes. The FRET signal could be diminished by the further addition of (unlabeled) RRM2, no doubt reflecting competition for binding to the i-motif DNA. These experiments using the individual RRM domains from hnRNP LL confirm the role of this transcription factor in activation of BCL2 transcription via the i-motif in the promoter element.