Lothstein L, Arenstorf H P, Chung S Y, Walker B W, Wooley J C, LeStourgeon W M
J Cell Biol. 1985 May;100(5):1570-81. doi: 10.1083/jcb.100.5.1570.
The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (Beyer, A. L., M. E. Christensen, B. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(B2). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein-RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1(C2).
海拉细胞40S异质核核糖核蛋白单颗粒的大部分蛋白质质量由六种蛋白质的多个拷贝组成，这些蛋白质在SDS凝胶中分离为三组双峰带（A1、A2；B1、B2；以及C1、C2）（拜尔，A.L.，M.E.克里斯蒂安森，B.W.沃克，和W.M.勒斯特尔金。1977年。《细胞》。11: 127 - 138）。我们在此报告，当40S单颗粒短暂暴露于核糖核酸酶时，蛋白质A1、C1和C2会随着大多数前体信使RNA序列的丢失而同时溶解。其余蛋白质以（A2）3（B1）的四聚体或（A2）3（B1）（B2）的五聚体形式存在。这些四聚体可能以高度特异性的方式重新缔合形成两种不同结构中的任何一种。在0.1M盐中，大约12个四聚体（源自三或四个单颗粒）重新组装形成高度规则的结构，这些结构可能具有十二面体对称性。这些结构在43S沉降，宽度为20 - 22nm，质量接近230万。这些结构含有450 - 500个缓慢标记的RNA碱基，这些碱基在凝胶中以200 - 220个碱基长度的片段形式迁移。在9mM盐中，四聚体重新缔合形成长度不确定、螺距接近60nm、直径接近18nm的2.0M盐不溶性螺旋丝。如果40S单颗粒用无核酸酶的蛋白酶短暂处理，被核酸酶溶解的相同蛋白质（A1、C1和C2）会被优先切割。这种蛋白质切割与大多数异质核RNA的解离有关。蛋白质A2和B1再次重新组装形成均匀的球形颗粒，但这些颗粒的沉降速度比完整的单颗粒略慢。这些发现表明，蛋白质A1、C1和C2以及大多数前体信使序列在完整的单颗粒中占据外围位置，并且它们的同型和异型缔合依赖于蛋白质 - RNA相互作用。蛋白质交联研究表明，A1、A2和C1的三聚体作为40S颗粒中最容易稳定的同型缔合存在。这支持了A和C蛋白与B蛋白的3:1比例（通过密度测定），并表明40S单颗粒由三或四个重复单元组成，每个单元包含3（A1）、3（A2）、1（B1）、1（B2）、3（C1）和1（C2）。
