Myers Jeffrey C, Moore Sheila A, Shamoo Yousif
Department of Biochemistry and Cell Biology, Rice University, 6100 S. Main Street, Houston, TX 77005, USA.
J Biol Chem. 2003 Oct 24;278(43):42300-6. doi: 10.1074/jbc.M306147200. Epub 2003 Aug 6.
Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(TTAGGG)n. Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG)2 is approximately 5 nm at 100 mm NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the Tm of the hTR sequence d(TAGGGT)4 from 67.0 degrees C to 36.1 degrees C at physiological conditions (150 mm KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.
异质性核糖核蛋白A1（hnRNP A1）是一种丰富的核蛋白，参与RNA加工、可变剪接和染色体维持。hnRNP A1可被蛋白水解为解旋蛋白（UP1），这是一种22.1 kDa的蛋白质，对富含嘌呤的单链核酸具有高亲和力，包括人类端粒重复序列（hTR）d(TTAGGG)n。以与hTR结合的UP1结构为指导，我们在DNA的两个位置之一掺入了荧光鸟嘌呤类似物6-MI，以促进结合研究。一个位置是6-MI与相邻嘌呤保持堆积，另一个位置是UP1结合后它完全解堆积。通过X射线晶体学确定了与UP1复合的两种修饰寡核苷酸的结构，以验证我们设计的有效性，并且已证明6-MI是在复合物形成时堆积环境发生变化的位置中用于单链核酸相互作用的出色报告分子。我们已经表明，在100 mM NaCl、pH 6.0条件下，UP1对d(TTAGGG)2的亲和力约为5 nM，并且我们对d(TTAGG(6-MI)TTAGGG)的结合研究表明，结合对盐和pH仅适度敏感。UP1还具有强大的G-四联体去稳定化活性，在生理条件（150 mM KCl，pH 7.0）下，将hTR序列d(TAGGGT)4的熔解温度从67.0℃降低到36.1℃。与X射线晶体学确定的结构一致，UP1能够在溶液中以二聚体形式结合hTR序列，并支持hnRNP A1在阵列中与核酸结合的模型，该阵列可能形成一组连续的反平行单链核酸结合裂隙。这些数据表明，hnRNP A1在可变剪接位点选择、RNA加工、RNA运输和染色体维持中看似不同的作用反映了其结合富含嘌呤的共有序列（nYAGGn）并使潜在有害的G-四联体结构去稳定化的能力。
