Liphardt J, Napthine S, Kontos H, Brierley I
Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.
J Mol Biol. 1999 May 7;288(3):321-35. doi: 10.1006/jmbi.1999.2689.
RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2.
RNA假结是参与多种生物过程的结构元件。在-1核糖体移码位点，已鉴定出几种类型的假结，它们在结构和功能上有所不同。在传染性支气管炎病毒（IBV）中发现的假结是那些具有11-12个碱基对的长茎1和长环2（30-164个核苷酸）的假结的典型代表。另一组可区分的假结包含仅5至7个碱基对的茎和较短的环。一种这样的假结，即小鼠乳腺肿瘤病毒（MMTV）的假结的核磁共振结构表明，它在茎1-茎2交界处发生扭结，并且这种扭结构象对于有效的移码至关重要。我们最近研究了在基于IBV的假结中调节茎1长度和稳定性对移码的影响，发现有效移码需要至少11个碱基对的茎1。在这里，我们描述了绕过对11个碱基对茎1的要求并将短的无功能的源自IBV的假结转化为高效的、扭结的移码假结所必需的序列操作。在茎1-茎2交界处简单插入一个腺嘌呤残基（扭结假结的一个基本特征）不足以产生功能性假结。还需要一个额外的变化：只有当环2的最后一个核苷酸从G变为A时，才能恢复有效的移码。环2末端需要一个A与类似于其他RNA三级结构中描述的环-螺旋接触一致。对所提出的相互作用的两个伙伴，即环2末端腺嘌呤残基和茎1顶部附近的两个G.C对进行的突变分析表明，这种相互作用对于有效的移码至关重要。当环2从8个核苷酸增加到14个核苷酸时，对3'-末端A残基的特定要求消失了，这表明环-螺旋接触可能仅在那些具有短环2的假结中是必需的。
