Name: an in vitro assay to detect trna-isopentenyl transferase activity
Uploaded: 2018-10-08T13:00:00
Description: Watch this Scientific Journal Video about An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity at JoVE.com

我们要感谢大卫 Engelke 博士对这篇手稿的指导和有益的评论。睡衣州立大学实验室启动基金;民建联1R15AI130950-01。

注: 该协议是根据参考文献24改编的。
1. 获得感兴趣的 RNA 和酵素
<ol><li>使用体外转录 rna 内部标记与32P, 重组 His6-Mod5 表达和纯化大肠杆菌, 如前所述24。<ol>
<li>用 T7 RNA 聚合酶在未标记 ATP 的存在下, 引入体外转录 rna (3 节), 双绞线, CTP, GTP 和10µCi 凝胶纯化, 乙醇沉淀α ATP, 悬浮 1x TE (10 毫米三盐酸 pH 7.5, 0.1 毫米 ED...

<ol>
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RNA 的修饰继续被证明在细胞和生物体功能中扮演越来越重要和不同的角色。因此, 研究 RNA 修饰酶的检测的发展对于更好地理解生物学的基本方面是至关重要的。本协议描述了一种高分辨率的体外检测, 以表征 Mod5 的 tRNA 修饰活性。
该协议具有提供直接且容易解释的 isopentenylation 读数的明显优势。所述协议允许对 isopentenyl 转移酶进行健壮的生化表征。此外, 该系统可用?...

已确定至少163种不同的转录后水平 RNA 修饰, 这些修改赋予 rna 不同的上下文相关功能, 直接影响 rna 结构, 并影响 rna 与其他分子1,2。随着对 rna 修饰的数量和种类的认识的增加, 重要的是开发能够可靠地审问 rna 修饰和催化它们的酶的化验。
第一个 RNA 修改被发现发生在基37在 tRNAs, 毗邻反密码子在 3 ' 边3,4。isopentenyl 组从 dimethylallylpyrophosphate (DMAPP) 转移到腺苷 37 (i6A37) 3,5的 N6 位置, 在细胞质和线粒体 tRNAs 的一个子集上。i6A37 提高翻译的保真度和效率通过增加 tRNA 的亲和性核糖体4,6和 i6A37 是重要的压力反应的细菌7。执行这种修饰的酶称为 tRNA isopentenyl 转移酶, 在细菌8、9、真菌10、蠕虫11、植物12和更高的真核生物13中高度保守., 包括人14。
人类 tRNA isopentenyl 转移酶基因TRIT1的突变与人类疾病有关。例如, TRIT1的突变与严重的线粒体疾病相关, 可能是由线粒体蛋白合成15、16的缺陷引起的。此外, TRIT1已被描述为肿瘤抑制基因17,18和涉及多种类型的癌症, 包括黑色素瘤19, 乳房20, 胃21, 肺癌22 ,23。最后, TRIT1 和 Mod5 (酿酒酵母) isopentenyl 转移酶是容易聚集的蛋白质, 形成朊蛋白样的淀粉样纤维24,25,26。这些观察可能牵连到神经退行性疾病中的 tRNA isopentenyl 转移酶, 尽管这方面的直接证据尚未显示出来。
考虑到 isopentenyl 转移酶在翻译和疾病中发挥的作用, 直接测量 i6isopentenyl 转移酶活性的方法对于在正常和疾病状态下对这些酵素的机械理解是重要的。越来越多的方法可用于检测 i6RNA 修饰, 包括体外isopentenylation 化验, 无 i6(PHA6) 化验, 薄层色谱 (TLC), 氨基酸的阳性杂交验收活动化验和质谱方法 (参考文献4)。
用14DMAPP 和未标记 tRNAs 对体外isopentenylation 进行了描述。在这项试验中, 放射性碳被 isopentenyl 转移酶从14DMAPP 的 RNA 转化为核糖核酸。虽然这种检测是高度敏感的, 通常很难确定的具体残留物, 修改9,20,27。PHA6 化验依赖于笨重的 i6的修改干扰杂交的32P 标记探针跨越改良残留。因此, 在没有 i6修改18,28,29的情况下, 杂交是更大的。PHA6 检测是高度敏感的, 能够分析从细胞裂解物提取的总 RNA。此外, 设计特定于感兴趣的 RNA 的探针的能力给这个方法坚固目标灵活性。然而, PHA6 化验仅限于对探针目标区域内残留物进行的修饰的表征, 因此不太可能发现新的修饰点。此外, 由于缺乏绑定指示修改, 其他影响 RNA 绑定的修改或突变将混淆数据分析。
另一种方法是将苄基 DEAE 纤维素 (BD) 纤维素色谱与氨基酸接受活性结合起来, 作为6tRNA30的一项修正。这种方法直接分析了 i6的功能修改, 但它是一种间接的方法来检测 i6的修改, 并缺乏解决方案, 以地图修改的特定残留在 RNA。采用薄层色谱法检测总 tRNA i6的修改。在这种方法中, 将内部32P 标记的 tRNAs 消化为单核苷酸, 并用二维薄层色谱分析来识别 isopentenylation。这种方法对于在给定的 RNA 样本中检测总 i6A 非常敏感, 但在消化时, 所有序列信息都丢失;因此, 调查员没有办法确定哪些残留物已被修改31。
最近, 液相色谱-串联质谱 (LC-ms) 方法已经发展, 定量比较的总 RNA 修改之间的物种, 细胞类型和实验条件32,33, 34。这种方法的一个局限性是, 它不太能确定在 RNA 中的身份和位置, 而改性核苷的来源是34。此外, 执行这些实验所需的专门知识和设备限制了这种方法的实用性。
此外, 还开发了几种下一代测序技术, 以绘制转录34的 RNA 修改图。免疫沉淀的 rna 与特定的修改 (RIP 序列) 的抗体, 使调查人员能够识别所有序列包含特定的修改35,36。此外, 基于逆转录酶的化学序列和非随机失配排序方法依赖于修正残留物37、38、39的反向转录反应的扰动。尽管这些技术的优势, 以地图 RNA 修改转录, RIP 序列和化学序列技术是有限的, 缺乏可靠的抗体或活性化学品, 每一个具体的修改, 分别为34。此外, 稳定的 RNA 结构可以阻碍执行化学序列和非随机失配测序技术所需的逆转录酶酵素。tRNAs 的高度修改和结构稳定的性质使他们特别难以用这些技术来审问。到目前为止, 基于下一代测序技术还没有被利用来映射 i6修改34。
在这里, 我们描述了一个简单和直接的方法来检测 i6tRNA 修改的体外。该方法利用 rna 的孵化与重组的酵母isopentenyl 转移酶 (Mod5), 其次是 RNase T1 消化, 1 维凝胶电泳分析映射 i6修改。这种方法是直接的, 需要很少的专门知识来分析数据。此外, 这种方法可以适应其他 rna 修饰酶, 包括酶, 共价键改变 rna 分子量或 rna 的流动性通过凝胶。

<table border="0" cellpadding="0" cellspacing="0" style="width:677px;" width="678">
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</table>

an in vitro assay to detect trna-isopentenyl transferase activity

n-6-isopentenyladenosine RNA 修饰在功能上是多样的, 在原核生物和真核生物中高度保守。其中最保守的 N6-isopentenyladenosine 修改发生在 A37 的位置在 tRNAs 的子集。通过增加 tRNA 对核糖体的亲和性, 这种修正提高了翻译效率和保真度。在真核生物中负责这种修饰的酶的突变与几种疾病状态有关, 包括线粒体功能障碍和癌症。因此, 了解这些酶的基底特异性和生物化学活性对了解正常和病理真核生物具有重要意义。使用了多种方法来描述 i6的修改。本文描述了 Mod5 检测 isopentenylation 的直接方法。该方法利用 rna 的孵化与重组 isopentenyl 转移酶, 其次是 RNase T1 消化, 1 维凝胶电泳分析, 以检测 i6的修改。此外, 还讨论了该协议对其他 RNA 修饰酶特性的潜在适应性。

Mod5 在 DMAPP 存在或不存在的情况下, 用酪氨酸 tRNA 或丝氨酸 tRNA 孵化。继改性反应后, 产品被 RNase T1-digested, 它会将所有 guanosines 的 3 ' 末端, 留下 3 ' 鸟嘌呤 monophosphates (GMP)24 (图 1)。对 rna 的完全消化产生一个可预测的模式核素片段 (图 2a), 然后解决了20% 聚丙烯酰胺变性凝胶。将 isopentenyl 组从 DMAPP ...

Watch this Scientific Journal Video about An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity at JoVE.com

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

在这里, 我们描述了一个协议的生化特性的酵母 rna 修饰酶, Mod5, 并讨论如何将该协议适用于其他 RNA 修饰酶。

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

该协议的总体目标是在振动中对tRNA-Isopentenyl转移酶活性进行生化特征。该方法对纯化重组酶的tRNA-isopentenyl转移酶活性的体外检测和表征非常有用。该技术的主要优点是，它是一种简单而直接的检测，用于检测共价RNA修饰。虽然这种方法为tRNA-isopentenyl转移酶活性提供了见解，但它对于广范围或RNA修改酶的生化特性具有潜在的适应性。本程序的第一步是彻底清洁用于铸造凝胶的玻璃板。用肥皂和水清洗盘子，用去离子水冲洗好，然后用异丙醇和无绒湿巾清洗盘子。接下来，组装板和空间。混合以下试剂，制造100毫升20%丙烯酰胺、7.5摩尔尿素、1x TBE凝胶。80毫升尿素凝胶浓缩物，10毫升尿素凝胶稀释剂，10毫升尿素凝胶缓冲液。在混合物中加入40微升T-med和800微升新鲜准备的10%铵。将凝胶溶液拉入大型注射器中，在玻璃板之间分配凝胶溶液，同时分配溶液以防止气泡形成。让凝胶在室温下凝固30分钟。凝胶凝固后，将凝胶种植到垂直凝胶装置上。用 1x TBE 填充上部和下部缓冲室。在加载凝胶前两小时，以 20 毫安预运行凝胶两小时，使缓冲边界超过最小的寡核苷酸。在17微升的最终体积中为RNA的开基化测定准备每种反应。添加到每个管50毫莫勒Tris-HCl， pH 7.2， 1.2 毫摩尔 ATP， 5.8 毫摩尔氯化镁， 0.2 毫摩尔 DMAPP， 10 单位 RNase 抑制剂， 40， 0 CPM 内部 32P 标记 RNA， 5.3 微摩尔 Mod5 和 1.2 毫摩尔 2-甲酸酯醇.在37摄氏度下孵育反应一小时。一小时后，乙醇使用2.5体积的100%乙醇和1/10体积的3.5摩尔醋酸钠pH5.5，将RNase沉淀为负20摄氏度，过夜一小时。接下来，将RNA样品在15，400克和4摄氏度下离心20分钟。小心地去除上一液，用 500 微升 70% 乙醇清洗每个 RNA 颗粒。将样品在15，400克和4摄氏度下离心5分钟。小心地去除上一液，将RNA颗粒干燥15分钟，或直到所有乙醇蒸发。接下来，将每个RNA颗粒重新悬浮在8个摩尔尿素的10微升中。在每个样品中加入150个单位的RNase T1，并在37摄氏度下孵育过夜。第二天，向每个样本添加两个 6 倍加载缓冲区的微升。在预运行、20%聚丙烯酰胺、7.5 摩尔尿素凝胶上加载每个RNA样品的10微升。以 25 毫安运行凝胶两小时。两小时后，停止电泳，将凝胶从仪器上取出。打破两个玻璃板之间的密封件，小心地拆下其中一个玻璃板，将凝胶留在任一板上。在凝胶上放置一层塑料包裹，在磷屏幕上暴露三个小时。该协议可靠地检测由S.cerevisiae异丙酶Mod5修改的规范和非核tRNA残留物的开模化。在此示例中，酪氨酸 tRNA 内部标有 32P 腺苷，Mod5 在存在与否时用 RNase 孵育。RNA与RNase T1一起消化，并在变性多丙烯酰胺凝胶上解析。Mod5 在 DMAPP 存在的情况下修改预测的残留物，并指示转移的 10 核苷酸，三重腺苷含有片段在核苷位置 36 到 38 包含酪氨酸 tRNA 中的片段。修改后的腺苷 37 驻留用星号表示。同样，当用Mod5和DMAPP孵育血清tRNA时，观察到10个核苷酸三重腺苷在核苷位置36至38位置含有片段的完整转移。有趣的是，观察到七核苷酸片段的部分移位，该片段不包含在核苷位置36至38时含有片段的三重腺苷，这表明在核苷位置36到38序列和结构上含有片段的三重腺苷可能不需要体外活动。一旦掌握了这项技术，如果执行得当，每天需要两到四到六个小时。尝试这个程序是重要的是要记住保持和监测RNA的完整性，因为RNA降解会影响RNA的修改，并混淆数据解释。按照这个程序突变研究使用你感兴趣的酶， 可以做确定特定的残留物或酶活性所需的域.在开发之后，这项技术为RNA生物学领域的研究人员铺平了道路，即原核酶和真核酶的tRNA-isopentenyl转移酶活性。看完这段视频后，您应该对如何执行检测您感兴趣的酶的 isopentenyl 转移酶活性的协议有一个很好的理解。不要忘记，使用放射性操作可能极其危险，在执行此过程时，应始终采取预防措施，例如穿着适当的 PPE、使用适当的屏蔽以及正确处理放射性。

Research

JoVE Journal

Biochemistry

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

A Fluorescence-based Assay of Phospholipid Scramblase Activity

磷脂Scramblase活动的基于荧光的测定法

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Quantification of Hypopigmentation Activity In Vitro

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

DetectSyn：一种快速、无偏倚的荧光方法，用于检测突触密度的变化

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

使用荧光测定法或流式细胞术测量半胱天冬酶活性

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...