Greatorex J S, Laisse V, Dockhelar M C, Lever A M
Department of Medicine, University of Cambridge, UK.
Nucleic Acids Res. 1996 Aug 1;24(15):2919-23. doi: 10.1093/nar/24.15.2919.
The formation of a genomic RNA dimer appears to be a critical step in the life cycle of all retroviruses. To investigate the site and nucleotide interactions involved in this process, a 531 bp DNA fragment encompassing sequences up- and downstream of the splice donor in human T cell leukaemia virus type 1 (HTLV-1) was inserted into a plasmid vector under the control of the SP6 promoter. RNA transcripts generated in vitro from this template formed dimers which could be dissociated by heating at 60-80 degrees C for 3 min. The physical properties of the dimeric RNA were not consistent with either Watson-Crick base pairing or guanine tetrad formation as being solely responsible for the interaction. Deletion mutagenesis identified a 32 nt sequence required for dimerisation. Computer modelling was carried out in order to identify putative RNA secondary structures within this essential region. A stem-loop structure was identified, the stem of which was conserved among different sequenced isolates of HTLV-1. This sequence also contains a 15 nt palindrome. We sought by disruptive and compensatory mutagenesis to define the possible roles of these two structures in dimer linkage.
基因组RNA二聚体的形成似乎是所有逆转录病毒生命周期中的关键步骤。为了研究这一过程中涉及的位点和核苷酸相互作用，将一段531 bp的DNA片段插入到受SP6启动子控制的质粒载体中，该片段包含人类1型T细胞白血病病毒（HTLV-1）剪接供体上下游的序列。从该模板体外产生的RNA转录本形成二聚体，可通过在60 - 80℃加热3分钟使其解离。二聚体RNA的物理性质与仅由沃森-克里克碱基配对或鸟嘌呤四联体形成负责相互作用的情况均不一致。缺失诱变确定了二聚化所需的32 nt序列。进行计算机建模以识别该关键区域内假定的RNA二级结构。鉴定出一种茎环结构，其茎在HTLV-1的不同测序分离株中是保守的。该序列还包含一个15 nt的回文序列。我们通过破坏性和补偿性诱变来确定这两种结构在二聚体连接中的可能作用。
