| Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins
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Author:
Date:
2017-03-20
[Abstract] Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from ...
[摘要] 核糖体失活蛋白（RIP）是由于其N-糖基化酶（EC 3.2.2.22）活性而不可逆地灭活核糖体的酶。酶从28S rRNA的腺嘌呤编号4324和大鼠核糖体中的核糖（或来自其他生物体的敏感核糖体中的等同腺嘌呤）切割N-糖苷键。该腺嘌呤位于α-原丝素 - 蓖麻毒素环（SRL）中，这对于在原核生物和真核生物中的mRNA-tRNA易位期间分别在核糖体上锚定延伸因子（EF）G和EF2至关重要。 RIP主要从植物中分离出来，这些蛋白质的实例是蓖麻毒蛋白或者口服抗病毒蛋白（PAP）。这些蛋白质，单独或作为免疫毒素的一部分，是癌症治疗的有用工具。以下方案描述了当通过在聚丙烯酰胺凝胶上电泳在来自兔网织红细胞裂解物的RIP处理的脱嘌呤RNA在酸性苯胺存在下孵育时检测释放的RNA片段的方法。发布的片段（Endo的片段）是RIP的动作的诊断。
Endo和Tsurugi首先在大鼠核糖体中描述了RIP对真核28S rRNA的N-糖基化酶活性，蓖麻毒素（Endo and ...
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| Northern Blot of tRNA in Yeast
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Author:
Date:
2013-04-05
[Abstract] tRNAs are small RNAs around 70-90 nt. tRNAs are different from many other small RNAs in that they are very abundant, which makes it difficult to study their transcriptional regulation by traditional northern blot. Traditional Northern blot involves incorporation of radioactive nucleotides through polymerization, however, tRNA is too short for polymerization. Traditional Northern blot detects changes in RNA levels, however, tRNA are so abundant that small changes in their levels will escape detection. For these reasons, metabolic labeling by radioactive Uracil has been used instead. However, ...
[摘要] tRNA是约70-90nt的小RNA。 tRNA与许多其他小RNA不同，因为它们是非常丰富的，这使得难以通过常规Northern印迹研究它们的转录调节。 传统的Northern印迹涉及通过聚合掺入放射性核苷酸，然而，tRNA对于聚合来说太短。 传统的Northern印迹检测RNA水平的变化，然而，tRNA是如此丰富，其水平的小变化将逃脱检测。 由于这些原因，已经使用了放射性尿嘧啶的代谢标记。 然而，代谢标记只能检查总tRNA的变化，但不能区分不同类型的tRNA。 以下方案描述了通过Northern印迹检查单个tRNA基因转录的方法。
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| Metabolic Labeling of Yeast RNA with Radioactive Uracil
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Author:
Date:
2013-04-05
[Abstract] To examine gene expression, Northern blot or Real-Time PCR can be used to detect low abundant RNA such as mRNA. However, for high abundant RNAs such as rRNA and tRNA, Northern blot will not be able to discriminate the newly synthesized RNA from total RNA. Therefore, metabolic labeling is necessary to evaluate the expression of rRNA and tRNA genes. In this protocol, I describe a step-by-step method for labeling yeast RNA with radioactive uracil and examine the synthesis of these high abundant RNAs.
[摘要] 为了检查基因表达，可以使用Northern印迹或实时PCR来检测低丰度RNA如mRNA。 然而，对于高丰度的RNA如rRNA和tRNA，Northern印迹将不能区分新合成的RNA与总RNA。 因此，代谢标记是必要的，以评估rRNA和tRNA基因的表达。 在这个协议，我描述了一个分步的方法，用放射性尿嘧啶标记酵母RNA，并检查这些高丰度RNA的合成。
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