Identification of nucleotide binding sites in the poliovirus RNA polymerase.
Identification of nucleotide binding sites in the poliovirus RNA polymerase.
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脊髓灰质炎病毒 RNA 聚合酶中核苷酸结合位点的鉴定。
DOI：
10.1021/bi00019a005
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Ehrenfeld,E
中科院分区：
文献类型：
--
作者：
Richards,OC;Hanson,JL;Schultz,S;Ehrenfeld,E
修订稿于 1995 年 3 月 13 日收到® 摘要：脊髓灰质炎病毒 RNA 聚合酶 (3DP01) 通过还原氧化的 NTP 蛋白复合物与 [32P] 核糖核苷三磷酸 (NTP) 交联。用溴化氰消化交联复合物，并通过反相 HPLC 分离所得肽。通过二次 HPLC 分级分离和/或用内切蛋白酶 Glu-C、TPCK-胰蛋白酶或 Asp-N 进行额外消化，然后再进行另一次 HPLC 分级分离，纯化 32 P 标记的肽。确定了主要[32P]-肽的N-末端序列，并通过SDS-聚丙烯酰胺凝胶电泳获得了这些肽的大致大小。在 3Dpo1 中发现了两个主要的 NTP 结合位点。一个位点位于 Asp-266 和 Met-286 之间；该片段中可能的结合残基是 Lys-276、Lys-278 或 Lys-283。第二个结合位点位于 Ala-57 和 Met-74 之间，其中 Lys-61 或 Lys-66 作为可能的结合残基。这些区域与已知的 HIV-1 逆转录酶结构的比对使我们能够预测 RNA 和 DNA 聚合酶活性位点裂缝附近存在的保守“指”子域中下游核苷酸结合位点的位置。 N 端核苷酸结合位点不包含在其他聚合酶中保守的区域内。从 RNA 模板合成 RNA 是 RNA 病毒用来复制其基因组的独特生化反应。该反应由一类酶（RNA 依赖性 RNA 聚合酶）催化，其生化机制和结构与功能关系尚不清楚。其他类别的多核苷酸聚合酶（DNA 依赖性 DNA 或 RNA 聚合酶；RNA 依赖性 DNA 聚合酶）结构分析的最新进展表明，所有这些酶彼此之间具有有限的相似性（Moras，1993）。已经鉴定出一些可能解释 RNA 或 DNA 底物特异性的结构元件（Ollis 等，1985；Kohlstaedt 等，1992；Sousa 等，1993；Delarue 等，1990），其他保守残基可能在催化活性中发挥作用。可用的序列信息表明病毒 RNA 依赖性 RNA 聚合酶将遵循这种模式 (Pochet al., 1989)。
Revised Manuscript Received March 13, 1995® abstract: Poliovirus RNA polymerase (3DP01) was cross-linked to [32P] ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP—protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified bysecondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK—trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS—polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpo1 were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotidebinding site in the conserved “fingers” subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.The synthesis of RNA from an RNA template is a unique biochemical reaction utilized by RNA virusesto replicate their genomes. The reaction is catalyzedby a class of enzymes, RNA-dependent RNA polymerases, whose biochemical mechanisms and structure—functionrelationships are not well understood. Recent progress in the structural analyses of other classes of polynucleotidepolymerases (DNA-dependent DNA or RNA polymerases; RNA-depend-ent DNA polymerase) has indicated that all of these enzymes share a limited resemblance to one another (Moras, 1993). Several structural elements have been identified that may account for RNA or DNA substrate specificity (Ollis et al., 1985; Kohlstaedt et al., 1992; Sousa et al., 1993; Delarue et al., 1990), and other conserved residues likely have rolesin catalytic activity. Available sequence information indicates that the viral RNA-dependent RNA polymerases will follow in this pattern (Pochet al., 1989).