Nadler S G, Merrill B M, Roberts W J, Keating K M, Lisbin M J, Barnett S F, Wilson S H, Williams K R
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.
Biochemistry. 1991 Mar 19;30(11):2968-76. doi: 10.1021/bi00225a034.
The 319-residue A1 heterogeneous nuclear ribonucleoprotein is the best studied of the group of major or core mammalian hnRNP proteins that bind pre-mRNA immediately following transcription. Circular dichroism studies suggest that binding of A1 and its proteolytic fragment, UP1 (residues 1-195), to nucleic acids results in an unstacking of the bases of poly(A). On the basis of poly[d(A-T)] and poly[r(A-U)] melting studies, both A1 and UP1 are helix-destabilizing proteins. Titrations of A1 and UP1 with poly(A), poly(U), and poly[d(T)] suggest that these two proteins do not bind with significant base specificity. A previous study indicated that A1, which contains a glycine-rich COOH terminus (residues 196-319) not present in UP1, binds cooperatively to polynucleotides while UP1 does not [Cobianchi et al. (1988) J. Biol. Chem. 263, 1063-1071]. Here we confirm this latter finding and demonstrate that the cooperativity parameter for A1 binding, which has a value of about 35 for binding to both single-stranded RNA and DNA, is insensitive to the NaCl concentration at least up to 0.4 M. In contrast to the cooperativity parameter, the occluded site size for A1 binding to RNA is salt dependent and increases from about 14 to 28 upon increasing the NaCl concentration from 25 to 250 mM. This variation in site size is best explained by assuming that A1 can interact with nucleic acids via at least two different binding modes. Both A1 and UP1 have higher affinity for single-stranded as opposed to double-stranded nucleic acids and bind preferentially to single-stranded RNA as compared to DNA. Comparative studies on the binding of A1 versus UP1 to poly[r(epsilon A)] demonstrate that in addition to cooperative protein/protein interactions, the glycine-rich COOH-terminal domain of A1 is also directly involved in protein/nucleic acid interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
319个氨基酸残基的A1异质性核核糖核蛋白是在转录后立即结合前体mRNA的主要或核心哺乳动物hnRNP蛋白组中研究得最为深入的。圆二色性研究表明，A1及其蛋白水解片段UP1（1 - 195位氨基酸残基）与核酸的结合会导致聚腺苷酸（poly(A)）碱基的解堆积。基于聚[d(A - T)]和聚[r(A - U)]的解链研究，A1和UP1都是螺旋不稳定蛋白。用聚腺苷酸（poly(A)）、聚尿苷酸（poly(U)）和聚[d(T)]对A1和UP1进行滴定表明，这两种蛋白没有明显的碱基特异性结合。先前的一项研究表明，A1含有UP1中不存在的富含甘氨酸的COOH末端（196 - 319位氨基酸残基），它能协同结合多核苷酸，而UP1则不能[科比亚尼奇等人（1988年），《生物化学杂志》263卷，1063 - 1071页]。在此我们证实了后一发现，并证明A1结合的协同参数，其与单链RNA和DNA结合时的值约为35，至少在高达0.4M的NaCl浓度下对NaCl浓度不敏感。与协同参数不同，A1与RNA结合的封闭位点大小依赖于盐，当NaCl浓度从25mM增加到250mM时，该位点大小从约14增加到28。假设A1可以通过至少两种不同的结合模式与核酸相互作用，就能最好地解释位点大小的这种变化。与双链核酸相比，A1和UP1对单链核酸具有更高的亲和力，并且与DNA相比，它们优先结合单链RNA。对A1与UP1结合聚[r(εA)]的比较研究表明，除了协同的蛋白质/蛋白质相互作用外，A1富含甘氨酸的COOH末端结构域也直接参与蛋白质/核酸相互作用。（摘要截于250词）
