Chemical shift mapping of the RNA-binding interface of the multiple-RBD protein sex-lethal.
Chemical shift mapping of the RNA-binding interface of the multiple-RBD protein sex-lethal.
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多重 RBD 性致死蛋白的 RNA 结合界面的化学位移图。
DOI：
10.1021/bi970830y
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Wemmer,DE
中科院分区：
文献类型：
--
作者：
Lee,AL;Volkman,BF;Robertson,SA;Rudner,DZ;Barbash,DA;Cline,TW;Kanaar,R;Rio,DC;Wemmer,DE
TheDrosophilaprotein Sex-lethal (Sxl) contains two RNP consensus-type RNA-binding domains (RBDs) separated by a short linker sequence. Both domains are essential for high-affinity binding to the single-stranded polypyrimidine tract (PPT) within the regulated 3‘ splice site of thetransformer(tra) pre-mRNA. In this paper, the effect of RNA binding to a protein fragment containing both RBDs from Sxl (Sxl-RBD1+2) has been characterized by heteronuclear NMR. Nearly complete (85−90%) backbone resonance assignments have been obtained for unbound and RNA-bound states of Sxl-RBD1+2. A comparison of amide1H and15N chemical shifts between free and bound states has highlighted residues which respond to RNA binding. The β-sheets in both RBDs (RBD1 and RBD2) form an RNA interaction surface, as has been observed in other RBDs. A significant number of residues display different behavior when comparing RBD1 and RBD2. This argues for a model in which RBD1 and RBD2 of Sxl have different or nonanalogous points of interaction with thetraPPT. R142(in RBD2) exhibits the largest chemical shift change upon RNA binding. The role of R142in RNA binding was tested by measuring theKdof a mutant of Sxl-RBD1+2 in which R142was replaced by alanine. This mutant lost the ability to bind RNA, showing a correlation with the chemical shift difference data. The RNA-binding affinities of two other mutants, F146A and T138I, were also shown to correlate with the NMR observations.
