McAfee J G, Shahied-Milam L, Soltaninassab S R, LeStourgeon W M
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
RNA. 1996 Nov;2(11):1139-52.
The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein's asymmetry, its large occluded site size, and its RNA folding activity.
hnRNP复合物的C蛋白四聚体以高协同性结合约150 - 230个核苷酸的RNA（McAfee J等人，1996年，《生物化学》35:1212 - 1222）。三个连续结合的四聚体将700个核苷酸长度的RNA折叠成一个19S的三角形中间体，该中间体在体外引发40S hnRNP组装（Huang M等人，1994年，《分子细胞生物学》14:518 - 533）。尽管一直认为C蛋白的共有RNA识别基序（RRM，第8 - 87位氨基酸残基）是RNA结合的主要决定因素，但我们在此报告，一个包含第1 - 115位氨基酸残基的重组构建体在生理离子强度（100 mM NaCl）下对RNA的亲和力非常低。此外，我们证明一个缺少共有RRM但包含第140 - 290位氨基酸残基的N端缺失构建体以足以解释完整蛋白结合时观察到的总自由能变化的亲和力结合RNA。与天然C蛋白一样，140 - 290构建体在溶液中是四聚体，并且在100 - 300 mM NaCl中以耐盐方式化学计量地结合RNA。N端截短变体的第140 - 179位氨基酸残基包含11个碱性和2个酸性氨基酸残基，而第180 - 207位氨基酸残基指定了一个指导二聚体组装的亮氨酸拉链基序。C蛋白50个氨基酸残基的羧基末端内的元件是四聚体组装所必需的。一个碱性区域后接一个亮氨酸拉链与DNA结合蛋白的碱性 - 亮氨酸拉链（bZIP）类的结构域组织相同。与其他含有RRMs和bZIP基序的蛋白质的序列同源性表明，第140 - 207位氨基酸残基代表一个保守的bZIP样RNA结合基序（命名为bZLM）。围绕刚性亮氨酸拉链结构域的四个高亲和力RNA结合位点的空间取向可能部分解释了C蛋白的不对称性、其大的封闭位点大小以及其RNA折叠活性。
