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Molecular mechanism of antizyme frameshifting

抗酶移码的分子机制

基本信息

批准号：
08458223
负责人：
MATSUFUJI Senya
金额：
$ 4.29万
依托单位：
Jikei University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

1. Two sequence elements on antizyme mRNA, a pseudoknot structure downstream to and a GC-rich region upstream to the frameshift site, were confirmed to be stimulatory signals for the frameshifting both in cell-free and animal cell culture systems. The upstream element was shown to interact with elongating ribosomes. These cis-acting elements also increased the efficiencies of other frameshift events such as viral -l frameshifting. However, the frameshiftstimulating effect of polyamines was not mediated by interaction between the cis-acting elements and polyamines. Certain recoding phenomena at the terminator codons such as +1 frameshifting on E.coli release factor 2 (RE2) signal and stop codon readthrough on viral signals were also enhanced by polyamines. The possibility that polyamines repress the termination process was not supported by experimental results. The polyamine-interacting site(s)for frameshift stimulation is to be determined in the future.2. Analysis of frameshift efficie … More ncy and polyamine contents in transfected mammalian cells which stably express the reporter construct revealed that the unbound polyamines are responsible for the stimulation of antizyme frameshifting in the cells.3. Some compounds which either stimulate or inhibit antizyme frameshifting were found.Agmatine was one of the stimulatory substances, agmatine and showed anti-proliferative effect through depletion of the cellular polyamines. Diamine derivatives which inhibit the frameshifting in competition with polyamines will be useful as molecular probes to search the polyamine-interacting site(s).4. Antizyme cDNAs of chicken and zebrafish were isolated. Alternative functions of new antizyme isoforms were also found. These results revealed that antizyme and its frameshifting are phylogenetically old and widely distribute.5. A fission yeast, Schizosaccharomyces pombe, was shown to be a suitable model organism for genetic screening of trans-acting factors involved in antizyme frameshifting. Construction of an assay system for polyamine-dependent antizyme frameshifting in S.pombe is underway. Less

1.在无细胞培养系统和动物细胞培养系统中，抗酶基因上的两个序列元件，即位于移码位点下游的假结结构和位于移码位点上游的富含GC的区域，被证实是移码的刺激信号。上游元件被证明与拉长的核糖体相互作用。这些顺式作用元件也提高了其他移码事件的效率，例如病毒L移码。然而，多胺的移码刺激作用并不是通过顺式作用元件和多胺之间的相互作用来实现的。在终止子密码子上的某些重新编码现象也被多胺增强，例如在大肠杆菌释放因子2(RE2)信号上的+1移码和在病毒信号上的终止密码子通读。实验结果不支持多胺抑制终止过程的可能性。结论1.移码刺激的多胺相互作用位点(S)有待进一步确定。移码效率…的分析在稳定表达报告构建体的哺乳动物细胞中，Ncy和多胺的含量较高，表明游离多胺负责刺激细胞内抗酶的移码。发现了一些能刺激或抑制抗酶移码的化合物，胍丁胺是其中的一种刺激性物质，它通过耗尽细胞内的多胺而显示出抗增殖作用。与多胺竞争抑制移码的二胺类化合物有望成为寻找多胺相互作用位点的分子探针(S)。分离了鸡和斑马鱼的抗酶基因。还发现了新的抗酶亚型的替代功能。这些结果表明，抗酶及其移码在系统发育上是古老的，并且分布广泛。裂解酵母是一种适合于抗酶移码相关反式作用因子遗传筛选的模式生物。多胺依赖的抗酶在S.pombe中移码的检测系统正在构建中。较少