Du Z, Giedroc D P, Hoffman D W
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, 78712, USA.
Biochemistry. 1996 Apr 2;35(13):4187-98. doi: 10.1021/bi9527350.
A 36-nucleotide RNA with a sequence corresponding to the 5' end region of the gene 32 mRNA of bacteriophages T2 and T6 was analyzed by one- and two-dimensional NMR methods. NMR results provide clear evidence that the RNA is folded into a pseudoknot structure with two coaxial stems connected by two loops, in a classic pseudoknot topology. The pseudoknot is unusual in that one of the loops consists of only one nucleotide, which spans the major groove of a seven base pair helical stem. Imino proton resonances indicate the hydrogen bonding pattern within the pseudoknot, and two-dimensional NOE spectra provide information that describes many of the structural features. The temperature dependence of the UV absorption and imino proton exchange rates provides insight into the stability of the pseudoknot. A three-dimensional model of the pseudoknot that is consistent with our NMR data is presented, and features that may be important for stabilizing the pseudoknot structure are discussed. A substantial number of other putative RNA pseudoknots described in the literature have sequences and topologies that appear to be related to the T2 and T6 pseudoknots. We propose that these RNAs may be members of a family of pseudoknots related by a similar structural motif, which we refer to as "common pseudoknot motif 1" or CPK1. The bacteriophage T2/T6 pseudoknot can be considered a structural model for the CPK1 family. The common features of the CPK1 pseudoknots are a stem 2 with six or seven base pairs, a loop 1 consisting of a single adenosine, and a variable length stem 1 and loop 2. The first "dangling" nucleotide at the 3' end of the molecule probably stabilizes stem 2. The CPK1 family includes several of the retroviral pseudoknots associated with mRNA frameshifting and readthrough. The work presented here describes the first detailed NMR analysis of an RNA pseudoknot with an entirely natural nucleotide sequence.
利用一维和二维核磁共振方法分析了一段36个核苷酸的RNA，其序列与噬菌体T2和T6基因32 mRNA的5'端区域相对应。核磁共振结果提供了明确的证据，表明该RNA折叠成一个假结结构，具有两个通过两个环连接的同轴茎，呈经典的假结拓扑结构。该假结的不同寻常之处在于其中一个环仅由一个核苷酸组成，该核苷酸跨越一个七碱基对螺旋茎的大沟。亚氨基质子共振表明了假结内的氢键模式，二维NOE谱提供了描述许多结构特征的信息。紫外吸收和亚氨基质子交换速率的温度依赖性提供了对假结稳定性的深入了解。提出了一个与我们的核磁共振数据一致的假结三维模型，并讨论了可能对稳定假结结构很重要的特征。文献中描述的大量其他假定的RNA假结具有与T2和T6假结相关的序列和拓扑结构。我们提出这些RNA可能是由类似结构基序相关的假结家族的成员，我们将其称为“常见假结基序1”或CPK1。噬菌体T2/T6假结可被视为CPK1家族的结构模型。CPK1假结的共同特征是有一个含六个或七个碱基对的茎2、一个由单个腺苷组成的环1以及一个可变长度的茎1和环2。分子3'端的第一个“悬垂”核苷酸可能稳定茎2。CPK1家族包括几个与mRNA移码和通读相关的逆转录病毒假结。本文介绍的工作描述了对具有完全天然核苷酸序列的RNA假结的首次详细核磁共振分析。
