Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2.
Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2.
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在白色念珠菌返回跨座子TCA2中控制GAG-POL基因表达。
DOI：
10.1186/1471-2199-8-94
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发表时间：
2007-10-25
影响因子：
--
通讯作者：
Stansfield I
中科院分区：
文献类型：
--
作者：
Forbes EM;Nieduszynska SR;Brunton FK;Gibson J;Glover LA;Stansfield I
在C.在白色念珠菌反转录转座子Tca 2中，gag和pol ORF被UGA终止密码子分开，UGA终止密码子的3'是潜在的RNA假结。目前还不清楚Tca 2 gag UGA密码子如何被绕过以允许pol表达。然而，在其他逆转录元件中，gag终止密码子的翻译通读可以由其侧翼序列（包括3'假结）指导。在Tca 2中，gag终止密码子侧翼序列直接翻译通读和gag-pol融合蛋白的合成的假设被测试。将来自Tca 2 gag-UGA-pol连接的序列（300 nt）插入酿酒酵母双报告基因构建体中融合的lacZ和荧光素酶（luc）基因之间。虽然在UGA的下游，luc表达，但其表达不受在lacZ的3'末端插入额外的终止密码子的影响。Luc的表达是由一个以前未知的小启动子活性的gag-pol连接区。共同的证据表明，连接序列本身不能指导UGA通读。利用C.白念珠菌中，该gag-pol连接启动子和Tca 2长末端重复序列（LTR）启动子的活性进行了比较。在这两个启动子中，只有LTR启动子被热休克诱导，这也触发反转录转座。Tca 2 pol蛋白，在C.白色念珠菌，以允许检测，也是热休克诱导，表明pol蛋白表达从gag-UGA-pol RNA。这是第一次证明LTR启动子指导Tca 2 pol蛋白表达，并且pol蛋白从gag-pol RNA翻译，因此需要终止密码子旁路的机制。然而，与大多数其他逆转录元件和病毒通读信号相反，直接gag UGA侧翼序列不足以直接终止S.酿酒酵母，表明非经典机制直接gag UGA旁路Tca 2。
In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot. The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA. This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.
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