Secondary and tertiary structure in the central domain of adenovirus type 2 VA RNA I.
The small (160 nt) adenovirus RNA, VA RNAI, antagonizes the activation of the cellular protein kinase PKR (also known as DAI), a key regulator of gene expression. VA RNA consists of two stems separated by a complex region, the central domain, that is essential for its function. A notable feature of the central domain is a pair of tetranucleotides, GGGU and ACCC, which are mutually complementary and phylogenetically conserved. To investigate their role in the structure and function of VA RNA, we generated three sets of mutations designed to disrupt the putative stem and to restore it with different nucleotides. Substitutions in either of the tetranucleotides abrogated VA RNA function in two independent PKR-based assays, demonstrating the importance of these sequences in vivo. Compensating mutants restored function, indicating that the two tetranucleotides pair in the cell, but all of the compensating mutants were less active than wild-type VA RNA. The effects of the mutations on RNA structure were probed by nuclease sensitivity analysis. Pronounced changes in two loops in the central domain correlated closely with the formation and disruption of the stem, suggesting that the tetranucleotide stem defines a critical element in the structure of the central domain through tertiary interactions with the two loops. A model for the central domain is presented that accommodates these findings and also accounts for the known sites of PKR interaction.
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Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Mathews
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D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D019892:eIF-2 Kinase
D012342:RNA, Small Nuclear; D012367:RNA, Viral; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D012260:Ribonucleases