Cui T, Porter A G
Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent.
Nucleic Acids Res. 1995 Feb 11;23(3):377-82. doi: 10.1093/nar/23.3.377.
We previously showed that encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) binds specifically to 3'-terminal segments of EMC virus RNA. This binding, which depends on both the 3'-noncoding region (3'-NCR) and 3'-poly (A) tail [together denoted 3'-NCR(A)], may be an important step in the initiation of virus replication. In this paper, the 3'-NCR and 3'-poly(A) were separately transcribed then mixed, but no complex with 3Dpol was obtained, showing that covalent attachment of the 3'-poly(A) to the 3'-NCR is essential for complex formation. Mutational and deletion analyses localized a critical determinant of 3Dpol binding to a U-rich sequence located 38-49 nucleotides upstream of the 3'-poly(A). Similar analyses led to the identification of a sequence of A residues between positions +10 and +15 of the 3'-poly(A) which are also critical for 3Dpol binding. As U-rich and A-rich regions are important for 3Dpol binding, a speculative model is proposed in which 3Dpol induces and stabilizes the base-pairing of the 3'-poly(A) with the adjacent U-rich sequence to form an unusual pseudoknot structure to which 3Dpol binds with high affinity.
我们先前表明，脑心肌炎（EMC）病毒的RNA依赖性RNA聚合酶（3Dpol）特异性结合EMC病毒RNA的3'末端片段。这种结合依赖于3'非编码区（3'-NCR）和3'聚腺苷酸尾（一起表示为3'-NCR(A)），可能是病毒复制起始的重要步骤。在本文中，分别转录3'-NCR和3'-聚腺苷酸，然后混合，但未获得与3Dpol的复合物，表明3'-聚腺苷酸与3'-NCR的共价连接对于复合物形成至关重要。突变和缺失分析将3Dpol结合的关键决定因素定位到位于3'-聚腺苷酸上游38-49个核苷酸处的富含U的序列。类似的分析导致鉴定出位于3'-聚腺苷酸+10至+15位之间的A残基序列，其对于3Dpol结合也至关重要。由于富含U和富含A的区域对3Dpol结合很重要，因此提出了一个推测模型，其中3Dpol诱导并稳定3'-聚腺苷酸与相邻富含U的序列的碱基配对，形成一种异常的假结结构，3Dpol以高亲和力结合该结构。
