| In vitro Assessment of RNA Polymerase I Activity
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Author:
Date:
2017-02-05
[Abstract] In eukaryotic cells transcriptional processes are carried out by three different RNA polymerases: RNA polymerase I which specifically transcribes ribosomal RNA (rRNA), RNA polymerase II which transcribes protein-coding genes to yield messenger RNAs (mRNAs) and small RNAs, while RNA polymerase III transcribes the genes for transfer RNAs and for the smallest species of ribosomal RNA (5S rRNA). This protocol describes an in vitro assay to evaluate the rRNA transcriptional activity of RNA polymerase I. The method measures the quantity of radiolabelled uridine 5’ triphosphate incorporated in ex novo synthesized rRNA molecules by RNA polymerase I, in optimal conditions for the enzyme activity and in the presence of a toxin, α-amanitin, which inhibits RNA polymerase II and III ...
[摘要] 在真核细胞中，转录过程由三种不同的RNA聚合酶：特异性转录核糖体RNA（rRNA）的RNA聚合酶I，转录蛋白质编码基因以产生信使RNA（mRNA）和小RNA的RNA聚合酶II进行转录，RNA聚合酶III转录转录RNA和最小核糖体RNA（5S rRNA）的基因。该方案描述了用于评价RNA聚合酶I的rRNA转录活性的体外实验方法。该方法测量了合并的rRNA分子中掺入的放射性标记的尿苷5'-三磷酸的量通过RNA聚合酶I，在酶活性的最佳条件和毒素α-amanitin存在下，其抑制RNA聚合酶II和III而不影响RNA聚合酶I（Novello和Stirpe，1970）。
背景 在真核细胞中，RNA聚合酶I转录位于核仁中的核糖体基因，产生45S rRNA前体分子。这些被处理以形成成熟的18S，5.8S和28S ...
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| Measurement of Nucleotide Triphosphate Sugar Transferase Activity via Generation of Pyrophosphate
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Author:
Date:
2015-04-20
[Abstract] Nucleotide triphosphate (NTP) transferases (EC. 2. 7. 7. X) transfer a nucleoside monophosphate moiety from NTP to another substrate. NTP sugar transferases form a large member of the NTP transferase. There are many variations for the substrate combination of the NTP sugar transferases. It is important to measure the precise enzymatic activity of such NTP sugar transferases by a simple and efficient method. In our method, we measure pyrophosphate as a byproduct of nucleotide diphosphate (NDP)-sugar generation using the pyrophosphate assay kit. The kit reagents include two enzymes that convert pyrophosphate to phosphate, and then phosphorolyze chromogenic substrate to allow color development at 360 nm (see details below). Thus, the NDP-sugar formation can be simply traced as production of ...
[摘要] 核苷三磷酸（NTP）转移酶（EC.2.7.7.X）将核苷单磷酸部分从NTP转移到另一底物。 NTP糖转移酶形成NTP转移酶的大成员。 NTP糖转移酶的底物组合有许多变化。重要的是通过简单有效的方法测量这种NTP糖转移酶的精确酶活性。在我们的方法中，我们使用焦磷酸测定试剂盒测量焦磷酸作为核苷酸二磷酸（NDP）糖产生的副产物。试剂盒试剂包括将焦磷酸盐转化为磷酸盐，然后磷酸化发色底物以允许在360nm显色的两种酶（见下文详述）。因此，NDP-糖形成可以简单地追溯为焦磷酸盐的产生，其通过在360nm处的吸光度监测。该方法对于测量包括NTP糖转移酶的各种产生焦磷酸的酶是可靠的和多用途的。
[原理和概述] NTP转移酶催化可逆反应如下：NTP +糖-1P - NDP-糖+ PPi
酶反应可以监测为无机焦磷酸盐（PPi）的产生。 EnzChek焦磷酸测定试剂盒（Molecular Probes，Life Technologies，Carlsbad，CA）包括两种酶和用于显色以定量焦磷酸的足够材料。无机焦磷酸酶（试剂盒中的组分E）将焦磷酸盐降解为磷酸盐。嘌呤核苷磷酸化酶（PNP，组分B）利用磷酸将生色底物2-氨基-6-巯基-7-甲基嘌呤核糖核苷（MESG，组分A）切割成核糖-1-磷酸和2-氨基-6-巯基-7 - ...
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