Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons provides evidence for central non-canonical base-pairs, large A-rich loops, and a terminal branch
Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons provides evidence for central non-canonical base-pairs, large A-rich loops, and a terminal branch
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DOI：
10.1006/jmbi.1996.0543
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发表时间：
1996-10-11
影响因子：
5.6
通讯作者：
Sanger, HL
中科院分区：
文献类型：
--
作者：
Gast, FU;Kempe, D;Sanger, HL
利用聚合酶链式反应和体外转录技术，获得了PSTVd致死变异体的线状(非环状)单位长度(+)链RNA分子，当接种到番茄上时，能够引发典型的病害症状。非变性凝胶电泳显示，这些转录本可以采用与环状PSTVd分子相同的两种构象，即快速排列的杆状结构和缓慢迁移的十字形结构。用核酸酶和硫酸二甲酯对两个末端标记转录本的棒状构象进行了探测，发现在溶液中它的右侧部分与计算机预测的完全相同。然而，在左边的部分，三个独特的特征可以得到证实。(1)在中心区域，UV可交叉连接的环是闭合的，因此包含非正则碱基对(“环E结构”)。(2)在25℃到37℃之间存在三个大的“预熔环”。最左边的一个富含A，在大多数类病毒中保守，与致病性有关。(3)在左侧末端有两个小的茎环，而不是未分枝的结构。这些发夹可以在所有的“大”类病毒中形成(大约300个核苷酸或更长)，从而将这些类病毒中保守的十二聚体GGUUCCUGUGGU放置在上螺旋和分支连接内。来自Iresine llark的一个大类病毒认为其中一个发夹，而所有的“小”类病毒(约300个核苷酸或更小)都缺乏这两个，在几个植物病毒卫星RNA和Newt卫星RNA中，GAUU(U)基序和十二聚体残基出现在由两个或三个发夹组成的等效结构中。利用铅和Tb诱导的RNA切割，发现了金属结合部位，大部分在环中。因此，探测ELF PSTVd RNA并与其他类病毒、类病毒和卫星RNA进行比较有助于我们了解这些小致病RNA的二级结构。(C)1996年学术出版社有限公司
Using PCR and in vitro transcription, linear (non-circular) unit-length (+)strand RNA molecules of a lethal PSTVd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. Non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular PSTVd molecules, namely a fast ting rod-like and a slowly migrating cruciform structure. The rod-like conformer of two end-labelled transcripts was probed with nucleases and dimethyl sulphate, revealing that in solution its right part is identical to computer prediction. In the left part, however, three unique features could be substantiated. (1) In the central region a UV-cross-linkable loop is closed and thus contains non-canonical base-pairs (''loop E structure''). (2) Three large ''pre-melting loops'' are present at 25 degrees to 37 degrees C. The structure of the leftmost one, which is A-rich and conserved in most viroids, correlates with pathogenicity. (3) Two small stem-loops instead of an unbranched structure are found at the left terminus. These hairpins can form in all ''large'' viroids (approximate to 300 nucleotides or longer), thus placing the dodecamer conserved among these viroids, GGUUCCUGUGGU, within the upper helix and the branch junction. A large viroid from Iresine larks one of these hairpins, whereas all ''small'' viroids (approximate to 300 nucleotides or smaller) lack both, In several plant virus satellite RNAs and the newt satellite RNA, the motif GAUUU(U) and dodecamer remnants appear in an equivalent structure comprising two or three hairpins. Using lead- and terbium-induced cleavage of the RNA, metal binding sites were found, mostly in loops. Thus, probing elf PSTVd RNA and comparison with other viroids, virusoids, and satellite RNAs contributes to our understanding of the secondary structure of these small pathogenic RNAs. (C) 1996 Academic Press Limited