Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells.
Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells.
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发表时间：
1979-10
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通讯作者：
M. Minks;D. West;S. Benvin;C. Baglioni
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作者：
M. Minks;D. West;S. Benvin;C. Baglioni
在干扰素处理的 HeLa 细胞提取物中添加双链 RNA (dsRNA) 会导致从 ATP 合成 2',8-oligo(A)，并导致核糖体相关蛋白 M, = 72,000 磷酸化。先前描述的测定用于研究激活这两种酶活性的 dsRNA 的结构要求。用多核苷酸磷酸化酶合成具有不同C/G比例的Poly(CG)。这些多核苷酸或者用聚(1)退火以形成错配的dsRNA，或者用核糖核酸酶T1消化以产生更小的多核苷酸。平均每 8 个核苷酸就有 1 个错配的聚合物无法激活 2',5'-oligo(A) 聚合酶和蛋白激酶，而每 45 个核苷酸就有 1 个错配的聚合物则完全具有活性。通过聚(CG)的T1消化获得的多核苷酸通过凝胶过滤分级成离散尺寸的聚合物。这些大小的多核苷酸与高分子量 Poly(1) 退火，并测定 2',5'-oligo(A) 聚合酶和蛋白激酶的激活。这些酶不能被含有短于 30 个核苷酸的聚 (C) 的 dsRNA 激活。使用含有长于 65 至 80 个核苷酸的聚 (C) 的 dsRNA 可获得最大激活。使用已知长度的聚（A）和聚（U）形成的 dsRNA 观察到类似的激活大小要求。这些结果表明，所研究的两种酶的激活需要一段相对较长的碱基对，且不受互补链之一的错配或不连续性的干扰。这些结构特征与之前显示的 dsRNA 诱导干扰素所需的结构特征相似。
Addition of double-stranded RNA (dsRNA) to extracts of interferon-treated HeLa cells results in the synthesis of 2’,8-oligo(A) from ATP and in the phosphorylation of a ribosome-associated protein of M, = 72,000. Previously described assays were used to investigate the structural requirements of dsRNA for the activation of these two enzymatic activities. Poly(CG) with different ratios of C/G was synthesized with polynucleotide phosphorylase. These polynucleotides were either annealed with poly(1) to form mismatched dsRNA or digested with ribonuclease T1 to produce smaller polynucleotides. Polymers with an average of one mismatch every eight nucleotides failed to activate the 2’,5’-oligo(A) polymerase and protein kinase, whereas polymers with a mismatch every 45 nucleotides were fully active. The polynucleotides obtained by T1 digestion of poly(CG) were fractionated by gel filtration into discrete size polymers. These sized polynucleotides were annealed with high molecular weight poly(1) and assayed for activation of 2’,5’-oligo(A) polymerase and protein kinase. These enzymes could not be activated by dsRNA containing poly(C) shorter than 30 nucleotides. Maximal activation was obtained with dsRNA containing poly(C) longer than 65 to 80 nucleotides. A similar size requirement for activation was observed with dsRNA formed with poly(A) and poly(U) of known length. These results indicate that a relatively long stretch of base pairs, uninterrupted by either a mismatch or a discontinuity in one of the complementary strands, is required for the activation of the two enzymes studied. These structural characteristics are similar to those previously shown to be required for the induction of interferon by dsRNA.