Analysis of adenovirus VA RNAI structure and stability using compensatory base pair modifications.
Adenovirus VA RNAs are short non-coding transcripts that assist in maintaining viral protein expression in infected cells. Six sets of mismatch and compensatory base pair mutants of VA RNA(I) were examined by gel mobility and RNA UV melting to assess the contribution of each structural domain to its overall structure and stability. Each domain of VA RNA(I) was first assigned to one of two apparent unfolding transitions in the wild-type melting profile. The Terminal Stem and Central Domain unfold in a single cooperative apparent transition with an apparent T(m) of approximately 60 degrees C. In contrast, the Apical Stem unfolds independently and with much higher apparent T(m) of approximately 83 degrees C. Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition. The effects of mismatch and compensatory mutations at five of the six sites on the RNA melting profile are consistent with proposed base pairing and provide further validation of the current secondary structure model. Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNA(I) Central Domain structure essential for PKR inhibitory activity was identified.
Coventry
Veronica K
VK
Faculty of Life Sciences, The University of Manchester, Manchester, M1 7DN, UK.
Conn
Graeme L
GL
D000256:Adenoviridae / Q000235:genetics*; D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D020871:RNA Stability; D022661:RNA, Untranslated / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry*; D013053:Spectrophotometry; D019892:eIF-2 Kinase / Q000378:metabolism
D022661:RNA, Untranslated; D012367:RNA, Viral; D019892:eIF-2 Kinase