Identification of cis and trans elements involved in the cell cycle regulation of multiple genes in Crithidia fasciculata.
Identification of cis and trans elements involved in the cell cycle regulation of multiple genes in Crithidia fasciculata.
复制标题
鉴定束状短膜藻中多个基因细胞周期调控所涉及的顺式和反式元件。
DOI：
10.1128/mcb.19.9.6174
复制
发表时间：
1999
影响因子：
5.3
通讯作者：
Ray,DS
中科院分区：
文献类型：
--
作者：
Mahmood,R;Hines,JC;Ray,DS
几个DNA复制基因的转录本，包括编码核复制蛋白A大亚基和动基体拓扑异构酶II的RPA 1和TOP 2基因，在锥虫Crithidia fasciculata的细胞周期中周期性地积累。在这些mRNA的5′非翻译区（UTR）存在一个八聚体共有序列，即RAGAAG，它是TOP 2和RPA 1转录物周期性积累所必需的，也是核因子与这些基因的5′ UTR RNA结合所必需的。我们在这里表明，插入多个（6个）拷贝的这个八聚体序列（6×八聚体）到报告基因的5′ UTR赋予其转录物周期性积累。竞争实验和紫外交联实验表明，6×八聚体RNA和TOP 25 ′ UTR RNA与相同的核因子结合。6×八聚体中的单核苷酸取代消除了RNA凝胶位移，也阻止了报告基因转录本的循环积累。使用6×八聚体RNA作为配体通过亲和色谱纯化的称为循环元件结合蛋白的蛋白质与含有野生型八聚体的RNA结合，而不与含有突变八聚体的RNA结合。这些结果定义了C中的一个小序列元件。fasciculatamRNA的细胞周期调控所需的，并报告了一个推定的调节蛋白，特异性结合这些元素的鉴定和纯化。
Transcripts of several DNA replication genes, including theRPA1andTOP2genes, encoding the large subunit of nuclear replication protein A and the kinetoplast topoisomerase II, accumulate periodically during the cell cycle in the trypanosomatidCrithidia fasciculata. An octamer consensus sequence, CAUAGAAG, present in the 5′ untranslated regions (UTR) of these mRNAs is required for periodic accumulation of theTOP2andRPA1transcripts and also for binding of a nuclear factor(s) to the 5′ UTR RNAs of these genes. We show here that insertion of multiple (six) copies of this octamer sequence (6× octamer) into the 5′ UTR of a reporter gene confers periodic accumulation on its transcript. Competition experiments and UV cross-linking studies show that the 6× octamer RNA andTOP25′ UTR RNA bind to the same nuclear factor(s). Single-nucleotide substitutions in the 6× octamer that abolish the RNA gel shift also prevent cyclic accumulation of the reporter gene transcript. A protein termed cycling element binding protein, purified by affinity chromatography using 6× octamer RNA as a ligand, binds to RNAs containing wild-type octamers and not to those with mutant octamers. These results define a small sequence element inC. fasciculatamRNAs required for their cell cycle regulation and report the identification and purification of a putative regulatory protein that binds specifically to these elements.