Localization of binding site for encephalomyocarditis virus RNA polymerase in the 3'-noncoding region of the viral RNA.
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.
Cui
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Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent.
Porter
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AG
D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004686:Encephalomyocarditis virus / Q000201:enzymology*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009690:Nucleic Acid Conformation; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification
D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D011061:Poly A; D012321:DNA-Directed RNA Polymerases