PURIFICATION AND NUCLEIC-ACID BINDING-PROPERTIES OF A FRAGMENT OF TYPE C1/C2 HETEROGENOUS NUCLEAR RIBONUCLEOPROTEIN FROM THYMIC NUCLEAR EXTRACTS
PURIFICATION AND NUCLEIC-ACID BINDING-PROPERTIES OF A FRAGMENT OF TYPE C1/C2 HETEROGENOUS NUCLEAR RIBONUCLEOPROTEIN FROM THYMIC NUCLEAR EXTRACTS
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DOI：
10.1021/bi00193a015
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发表时间：
1994-07-12
期刊：
影响因子：
2.9
通讯作者：
MODAK, MJ
中科院分区：
文献类型：
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作者：
AMRUTE, SB;ABDULMANAN, Z;MODAK, MJ
一种sds -聚丙烯酰胺凝胶电泳显示分子量为12000的单链核酸结合蛋白（C/F），最初被认为是小牛胸腺末端脱氧核苷酸转移酶（TdT） AB形式的12 kda α亚基，已被纯化并鉴定为C1/C2型hnRNP蛋白的片段。根据nh2末端测序和质谱分析，C/F在C1/C2型hnRNP蛋白中含有94个类似的残基，从残基9到残基102左右。C/F可能是在细胞破坏后通过C1/C2 hnRNP型蛋白的有限蛋白水解在体外产生的。由于C/F几乎完全对应于许多真核RNA结合蛋白共享的类似于90残基的保守核糖核蛋白结合域（RBD），因此它为更好地表征C1/C2型hnRNP蛋白的结构域结构以及比较C1/C2型和A1型RNA结合域的核酸结合特性提供了机会[见Shamoo et al. (1994) Biochemistry，本刊上一篇论文]。与A1型RBD一样，C1/C2型RBD具有6-7个核苷酸的明显闭塞位点。C1/C2型RBD与同多核苷酸非合作结合，对RNA和单链而非双链核酸具有优先亲和力。C1/C2型RBD对RNA的亲和力比A1型RBD高100倍，这种亲和力的增加部分是由于额外的离子相互作用。后者约占C1/C2型RBD结合自由能的50%。而C1/C2型hnRNP蛋白在体内以非常紧密的四聚体形式存在，其结构为（C1）(3)C2 [Barnett et al. (1989) Mel。细胞。生物学报，9,492-498]，分离得到的C1/C2型RBD为单体。因此，四聚化的决定因素似乎位于C1/C2型RBD之外。苯丙氨酸19被确定为C1/C2型RBD与[d(T)]光化学交联的唯一点(8)。该残基对应于A1 RBD交联的主要位点[d(T)](8) [Merrill， B. M., Stone， K. L., Cobianchi， F., Wilson， S. H., and Williams， K. R.(1988)]。化学学报，2003,26(3):387 - 387。因此，尽管A1型和C型RNA结合域之间的直接序列一致性总体程度仅为17%，但这两种蛋白质似乎共享拓扑相似的RNP：寡核苷酸界面，当将其置于多结构域hnRNP蛋白的环境中时，该界面似乎不会发生实质性改变。
A single-strand nucleic acid binding protein (C/F) that has an apparent molecular weight of 12 000 on SDS-polyacrylamide gel electrophoresis and that was originally thought to be the 12-kDa alpha-subunit of the AB form of terminal deoxynucleotidyl transferase (TdT) from calf thymus has been purified and identified as a fragment of the type C1/C2 hnRNP proteins. On the basis of NH2-terminal sequencing and mass spectrometric analysis, C/F contains similar to 94 residues and spans from residue 9 to approximately residue 102 in the type C1/C2 hnRNP proteins. C/F is presumably produced in vitro via limited proteolysis of the type C1/C2 hnRNP proteins following cell disruption. Since C/F corresponds almost exactly to the similar to 90-residue conserved ribonucleoprotein binding domain (RBD) that is shared by many eukaryotic RNA binding proteins, it provided an opportunity to better characterize the domain structure of the type C1/C2 hnRNP proteins and to compare the nucleic acid binding properties of the type C1/C2 and A1 [see Shamoo et al. (1994) Biochemistry, preceding paper in this issue] RNA binding domains. Like the type A1 RBD, the type C1/C2 RBD has an apparent occluded site size of 6-7 nucleotides. The type C1/C2 RBD binds non-cooperatively to homopolynucleotides and has preferential affinity for RNA and for single as opposed to double-stranded nucleic acids. The type C1/C2 RBD has about a 100-fold higher affinity than the type A1 RBD does for RNA and some of this increased affinity results from additional ionic interactions. The latter account for similar to 50% of the free energy of binding of the type C1/C2 RBD. While the type C1/C2 hnRNP proteins exist in vivo as a very tight tetramer with the structure (C1)(3)C2 [Barnett et al. (1989) Mel. Cell. Biol. 9, 492-498], the isolated type C1/C2 RBD is a monomer. Hence, the determinants for tetramerization appear to lie outside the type C1/C2 RBD. Phenylalanine 19 was identified as the only point of photochemical cross-linking of the type C1/C2 RBD to [d(T)](8). This residue corresponds to the major site of cross-linking of the A1 RBD to [d(T)](8) [Merrill, B. M., Stone, K. L., Cobianchi, F., Wilson, S. H., and Williams, K. R. (1988) J. Biol. Chem. 263, 3307-3313]. Thus, even though the overall extent of direct sequence identity between the type A1 and C RNA binding domains is only 17%, both proteins appear to share a topologically similar RNP:oligonucleotide interface which does not appear to be substantially altered when it is placed within the context of a multidomain hnRNP protein.