Purvis I J, Bettany A J, Loughlin L, Brown A J
Institute of Genetics, University of Glasgow, UK.
Nucleic Acids Res. 1987 Oct 12;15(19):7951-62. doi: 10.1093/nar/15.19.7951.
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.
在携带于着丝粒质粒上的酵母丙酮酸激酶（PYK）基因的3'非翻译区（3'UTR）内构建了一个53个碱基对的缺失。使用pUC13多克隆位点DNA的各种模块化组件（单个单元=44 bp）来替换缺失区域，并在酵母中确定这些修饰对转录本稳定性和翻译的影响。基于特定寡核苷酸与pUC13多克隆位点或未改变的PYK 3'UTR序列的杂交，采用差异探测策略，能够区分突变型（质粒携带）和野生型（染色体）PYK转录本。在任何构建体中，mRNA稳定性均无显著改变，但3'UTR的截短或3'UTR中存在强发夹环结构会严重抑制PYK mRNA的翻译。N端编码序列中的一个特定突变，在一个3'“标记”的PYK质粒和一个PYK/LacZ融合基因中都产生了一个提前终止密码子，导致它们大多数转录本的翻译终止，但它们的化学半衰期未改变。这一观察结果与一些先前发表的数据（Losson & Lacroute（1979年）《美国国家科学院院刊》76，5134；Pelsey & Lacroute（1984年）《当代遗传学》8，277）不一致，但与我们自己早期的观察结果一致，即在酵母中核糖体负载与mRNA稳定性之间没有明显联系（Santiago等人（1986年）《核酸研究》14，8347）。讨论了这种差异的可能原因。
