HYDROGEN-BONDING CONTACTS IN THE MAJOR GROOVE ARE REQUIRED FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT PROTEIN RECOGNITION OF TAR RNA
HYDROGEN-BONDING CONTACTS IN THE MAJOR GROOVE ARE REQUIRED FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT PROTEIN RECOGNITION OF TAR RNA
复制标题
DOI：
10.1006/jmbi.1993.1129
复制
发表时间：
1993-03-05
影响因子：
5.6
通讯作者：
GAIT, MJ
中科院分区：
文献类型：
--
作者：
HAMY, F;ASSELINE, U;GAIT, MJ
通过制备一系列进行位点特异性官能团修饰的模型RNA底物，分析了TAR RNA的结合部位。测试RNA是通过对两个合成的短寡核苷酸进行热处理而制备的，形成具有富含U的凸起和与TAR RNA相同的侧翼序列的双重结构，与这些双链RNA的亲和力大约是野生型TAR RNA的一半。在位置U23或U25用碱基模拟物替代。O4-甲基-DT的N3位氢键能力不足，亲和力降低20倍以上。还测试了对TAR RNA茎中的嘌呤进行的修饰，这些修饰会影响双链RNA的主要或次要凹槽中的氢键能力。G26的N7位或A27的N7位的氮原子的去除亲和力为10-20倍。相比之下，用肌苷取代G26位小槽中的外环氨基对结合没有显著影响。A22和U23之间磷酸键上的单一甲基膦取代也会导致总结合能力的显著丧失，而富U凸起中的所有其他甲基膦取代都不会损害总结合。我们得出结论，这在TAR RNA分子的主槽中显示了一系列分散的位置，形成了多个特定的氢键。包括U23的N3-H、G26的N7、A26的N7和A22至U23之间的磷酸盐。
The binding site fortaton TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA.Tatbinds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23or U25by the base analogue. O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3position reducestataffinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7position of G26or the N7position of A27reducestataffinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affecttatbinding significantly. A single methylphosphonate substitution at the phosphate bond between A22and U23also leads to a significant loss oftatbinding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful totatbinding. We conclude thattatforms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7of G26, the N7of A26and the phosphate between A22and U23.