Regulation of RNA degradation by RNase

在S-腺苷蛋氨酸存在下，拟南芥的胱硫醚-γ-合成酶(CGS)基因在体外翻译时，Ser-94发生翻译停滞，导致CGS基因的降解。通过体外翻译的方法制备了携带CGS第一外显子编码区的mRNA，并将其用于小麦胚芽提取物的体外翻译。核糖体在转位阶段停滞不前，翻译中间产物肽基-tRNA位于A位。鉴定出一系列的mRNA降解中间产物，它们的5‘端相隔约30个核苷酸。有证据表明，这些降解中间体对应于堆积在最初停滞的核糖体后面的核糖体。据报道，Poly(G)能抑制小麦胚芽提取物体外翻译系统中的核糖核酸酶活性。当将Poly(G)加入到翻译反应混合物中时，CGS mRNA的5‘端片段的3’端与降解中间体的5‘端之一相匹配。结果表明，tRNAs的mRNA降解是通过内切核糖核酸酶Colicin E5进行的。Colicin E5优先消化谷氨酸二核苷酸。研究了结肠素E5的结构与底物专一性的关系。底物专一性可以用底物口袋的结构约束来解释。分析了能消化tRNA(Arg)的粘附素D在芽殖酵母和HeLa细胞中的表达效果。

When cystathionine gamma-synthase (CGS) mRNA of Arabidopsis is translated in vitro in the presence of S-adenosylmethionine (SAM), translation arrest is induced at Ser-94, which then induces CGS mRNA degradation. mRNA that carries the first exon coding region of CGS was prepared by in vitro translation and was used to program in vitro translation of wheat germ extract. Ribosome was stalled at the step of translocation and the peptidyl-tRNA, the translation intermediate, was located in the A-site. A series of mRNA degradation intermediates, whose 5' ends were separated by about 30 nucleotides from each other, were identified. Evidence was found that shows that these degradation intermediates correspond to ribosomes that were stacked behind the initial stalled ribosome. Poly (G) has been reported to inhibit RNase activity in the in vitro translation system of wheat germ extract. When Poly(G) was added to the translation reaction mixture, a 5'-fragment of CGS mRNA whose 3' end matches with one of the 5' -ends of the degradation intermediate. The result suggested that the mRNA degradation occurs by an endoribonucleolytic digestion.An Escherichia coli ribonuclease colicin E5 digests the anticodon loop of tRNAs for Trp, His, Asn and Asp. Colicin E5 preferentially digests GU dinucleotide. Structure and substrate specificity relationship of colicin E5 were studied. The substrate specificity could be explained by the structural constraints at the substrate pocket. Effects of expression of colicin D that digests tRNA(Arg) in budding yeast and HeLa cells were analyzed.