Coventry Veronica K, Conn Graeme L
Faculty of Life Sciences, The University of Manchester, Manchester, M1 7DN, UK.
Nucleic Acids Res. 2008 Mar;36(5):1645-53. doi: 10.1093/nar/gkn020. Epub 2008 Feb 3.
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.
腺病毒VA RNA是短的非编码转录本，有助于维持感染细胞中的病毒蛋白表达。通过凝胶迁移和RNA紫外熔解分析，对VA RNA（I）的六组错配和补偿性碱基对突变体进行了检测，以评估每个结构域对其整体结构和稳定性的贡献。首先将VA RNA（I）的每个结构域分配到野生型熔解曲线中两个明显的解折叠转变之一。末端茎和中央结构域在一个单一的协同明显转变中解折叠，表观熔解温度（T(m)）约为60℃。相比之下，顶端茎独立解折叠，表观T(m)约为83℃，明显更高。值得注意的是，该结构域在RNA中似乎表现为一个几乎完全自主的单元，反映了RNA在PKR结合和抑制之间的功能划分。六个位点中的五个位点的错配和补偿性突变对RNA熔解曲线的影响与提出的碱基配对一致，并进一步验证了当前的二级结构模型。在PKR抑制试验中测试了中央结构域中的突变，并确定了VA RNA（I）中央结构域中对PKR抑制活性至关重要的一个组成部分。
