Samuelsson T
Department of Medical Biochemistry, University of Göteborg, Sweden.
Nucleic Acids Res. 1991 Nov 25;19(22):6139-44. doi: 10.1093/nar/19.22.6139.
We have previously purified and characterized two different S. cerevisiae enzymes that produce pseudouridine specifically in nucleotide positions 13 and 55, respectively, in their tRNA substrates. The interactions of these enzymes with fluorinated tRNAs have now been studied. Such RNAs were produced by in vitro transcription using as templates synthetic genes that encode variants of a yeast glycine tRNA. RNAs substituted with fluorouracil were found to markedly inhibit pseudouridine synthase activity and the inhibitory effect of a tRNA was to a large extent dependent on the presence of fluorouracil in the nucleotide position where normally pseudouridylation occurs. Pseudouridine synthases were shown to form highly stable, non-covalent complexes with fluorinated tRNAs and we demonstrate that this interaction may be used to further characterize and purify these enzymes. The use of 5-fluorouracil as a cancer therapeutic agent is discussed in relation to our results.
我们之前已经纯化并鉴定了两种不同的酿酒酵母酶，它们分别在其tRNA底物的核苷酸位置13和55处特异性地产生假尿苷。现在已经研究了这些酶与氟化tRNA的相互作用。此类RNA是通过体外转录产生的，使用编码酵母甘氨酸tRNA变体的合成基因作为模板。发现用氟尿嘧啶取代的RNA会显著抑制假尿苷合酶活性，并且tRNA的抑制作用在很大程度上取决于正常发生假尿苷化的核苷酸位置中氟尿嘧啶的存在。假尿苷合酶显示与氟化tRNA形成高度稳定的非共价复合物，并且我们证明这种相互作用可用于进一步表征和纯化这些酶。结合我们的结果讨论了5-氟尿嘧啶作为癌症治疗剂的用途。
