A "slide-back" mechanism for the initiation of protein-primed RNA synthesis by the RNA polymerase of poliovirus
A "slide-back" mechanism for the initiation of protein-primed RNA synthesis by the RNA polymerase of poliovirus
复制标题
DOI：
10.1074/jbc.m307441200
复制
发表时间：
2003-11-07
影响因子：
4.8
通讯作者：
Wimmer, E
中科院分区：
文献类型：
--
作者：
Paul, AV;Yin, J;Wimmer, E
当UMP分子与末端蛋白VP g中酪氨酸的羟基共价连接时，脊髓灰质炎病毒RNA复制启动。该反应可通过使用两种纯化病毒蛋白（RNA聚合酶3D（pol）和病毒蛋白3CD（pro））、合成VPg、UTP和Mg 2+的测定法在体外重现。该反应的模板是脊髓灰质炎病毒RNA或位于蛋白2C（ATP酶）编码序列中的小RNA发夹（称为cre（2C））的转录物。反应产物为VPgpU和VPgpUpU，即3D（pol）用于RNA合成的引物。在该测定中，使用突变模板RNA，我们确定了精确的起始位点。我们的结果表明：1）3Dpol对它与VPg连接的核苷酸不具有严格的特异性，2）环中保守的（1）GXXXAAAXXXXXXA（14）序列的A-5是第一和第二UMP与VPg连接的模板核苷酸，3）VPgpUpU通过“滑回”机制合成，和4）A-6在回滑步骤期间为反应提供特异性，并且还调节尿苷酰化反应。在另外的实验中，我们确定了cre（2C）的（5）AAA（7）序列突变对病毒生长、RNA复制和2CATALYST蛋白活性的影响。此外，我们观察到G-1和A-5之间的间距和环的大小影响产量，但不影响VPg连接产物的性质。
Poliovirus RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction can be reproduced in vitro with an assay that utilizes two purified viral proteins, RNA polymerase 3D(pol) and viral protein 3CD(pro), synthetic VPg, UTP, and Mg2+. The template for the reaction is either poliovirus RNA or transcripts of a small RNA hairpin, termed cre(2C), located in the coding sequence of protein 2C(ATPase). The products of the reaction are VPgpU and VPgpUpU, the primers used by 3D(pol) for RNA synthesis. With mutant template RNAs in this assay we determined the precise initiation site. Our results indicate that 1) 3Dpol does not possess strict specificity toward the nucleotide it links to VPg, 2) A-5 of the conserved (1)GXXXAAAXXXXXXA(14) sequence in the loop is the template nucleotide for the linkage of both the first and second UMPs to VPg, 3) VPgpUpU is synthesized by a "slideback" mechanism, and 4) A-6 provides specificity to the reaction during the slide-back step and also modulates the uridylylation reaction. In additional experiments we determined the effect of mutations in the (5)AAA(7) sequence of cre( 2C) on viral growth, RNA replication, and on the activity of the 2CATPase protein. Furthermore, we observed that the spacing between G-1 and A-5 and the size of the loop affect the yield but not the nature of the VPg-linked products.