The effects of alterations within the 3' untranslated region of the pyruvate kinase messenger RNA upon its stability and translation in Saccharomyces cerevisiae.
The effects of alterations within the 3' untranslated region of the pyruvate kinase messenger RNA upon its stability and translation in Saccharomyces cerevisiae.
复制标题
丙酮酸激酶信使 RNA 3 非翻译区域内的改变对其在酿酒酵母中的稳定性和翻译的影响。
DOI：
10.1093/nar/15.19.7951
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发表时间：
1987
影响因子：
14.9
通讯作者：
A. Brown
中科院分区：
文献类型：
--
作者：
I. Purvis;A. Bettany;L. Loughlin;A. Brown
在着丝粒质粒上携带的酵母丙酮酸激酶（PYK）基因的3'非翻译区（3' UTR）内构建53个碱基对缺失。使用pUC 13多接头DNA（单个单元= 44 bp）的各种模块化组件来替换缺失的区域，并在酵母中确定这些修饰对转录物稳定性和翻译的影响。基于特定寡核苷酸与pUC 13多聚接头或未改变的PYK 3' UTR序列的杂交，使用差异探测策略可以区分突变体（质粒携带）和野生型（染色体）PYK转录本。在构建体中没有mRNA稳定性的任何显著改变，但是PYK mRNA的翻译由于3 ' UTR的截短或3' UTR中存在强发夹环结构而严重减少。在N-末端编码序列中的特定突变，其在3' '标记' PYK质粒和PYK/LacZ融合基因中产生提前终止密码子，中止了它们的大部分转录物的翻译，但使它们的化学半衰期不变。这一观察结果与以前公布的一些数据不一致（Losson & Lacroute（1979）Proc Natl Acad Sci USA 76，5134; Pelsey和Lacroute（1984）Curr Genet 8，277），但与我们自己早期的观察一致，即酵母中核糖体装载和mRNA稳定性之间没有明显的联系（圣地亚哥等（1986）核酸研究14，8347）。这种差异的可能原因进行了讨论。
A 53 basepair deletion was constructed within the 3' untranslated region (3' UTR) of the yeast pyruvate kinase (PYK) gene borne upon a centromeric plasmid. Various modular assemblies of the pUC13 polylinker DNA (single unit = 44 bp) were used to replace the deleted region, and the effects of these modifications upon both transcript stability and translation ascertained in yeast. The use of a differential probing stratagem, based on the hybridisation of specific oligonucleotides to either pUC13 polylinker or unaltered PYK 3' UTR sequences, allowed for discrimination between mutant (plasmid borne) and wild-type (chromosomal) PYK transcripts. In no construct was there any significant alteration in mRNA stability, but translation of the PYK mRNA was severely curtailed by truncation of the 3' UTR or the presence of a strong hairpin-loop structure in the 3' UTR. A specific mutation in the N-terminal coding sequences, which created a premature termination codon in both a 3' 'tagged' PYK plasmid and a PYK/LacZ fusion gene, aborted the translation of a majority of their transcripts but left their chemical half-lives unaltered. This observation is at variance with some previously published data (Losson & Lacroute (1979) Proc Natl Acad Sci USA 76, 5134; Pelsey & Lacroute (1984) Curr Genet 8, 277), but is consistent with our own earlier observation that there is no obvious link between ribosome loading and mRNA stability in yeast (Santiago et al. (1986) Nucleic Acids Res 14, 8347). Possible reasons for this disparity are discussed.