Name: dual dna rulers to study the mechanism of ribosome translocation with single-nucleotide resolution
Uploaded: 2019-07-08T13:00:00
Description: Watch this Scientific Journal Video about Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution at JoVE.com

这项工作得到了美国国家卫生研究院(R01GM111452,Y.W.,S.X.)的支持。Y. W. 感谢韦尔奇基金会(E-1721)的支持。

1. 核糖体复合物的制备
<ol><li>制作 1,000 mL 的 TAM10 缓冲液,包括 20 mM tris-HCl (pH 7.5)、10 mM Mg (OAc) 2、30 mM NH4Cl、70 mM KCl、5 mM EDTA、7 mM BME(2-mercaptoto 乙醇)和 0.05% 补间20。</li>
	<li>准备表1中列出的五种混合物。 注:核糖体来自MRE600菌株11。EF-Tu:伸长系数热不稳定。EF-T:伸长系数热稳定。GTP:三磷酸三磷酸二磷酸二苯基苯基。PEP:磷(苯酚)丙酮酸盐。</li>
	<li>在...

<ol>
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在我们的双标测定中,磁珠起着两个重要的角色。首先,它们充当力传感器,因为离心力与其浮力成正比。其次,磁珠是原子磁力计探测到的信号载体,这是目前最精确的磁传感器。结合机械操作和磁检测,FIRMS技术能够基于其关键的解散力(DNA标尺的基础)解决大量的分子相互作用。双标尺测定特别适用于在易位期间探测 mRNA 的两侧。因为它是一种仅依赖于双面的形成的物理方法,它不受 mRNA 边或其他生物?...

生物分子位移是研究相关生物函数机理的基本参数。一个特别的例子是核糖体易位1,2,在此期间,核糖体移动正好三个核苷酸 (nt) 在信使RNA (mRNA) 正常, 和一,2,或其他数字的nt,除了三个在帧移位。因此,需要分子标尺系统单nt分辨率来区分不同的步长大小。更大的挑战是探测入口和出口侧的核糖体运动。换句话说,只有使用双标尺系统,我们才能揭示mRNA是否平滑地穿过核糖体,或者有中间步骤,其中双方具有不同的步长大小,导致在核糖体。
已经开发出几种方法来解决解决核糖体出口侧不同步骤的第一个挑战(mRNA的3'末端)。双荧光素酶测定通过测量产生的不同蛋白质3,4的比例来解决不同的阅读帧。它仅适用于 mRNA 的 3' 端,因此不足以提供易位的完整图像。质谱法可以分析不同的肽片段,作为相应代码重新排列的结果5。但它不能确定核糖体在mRNA上移动多少nt。脚趾印刷测定是另一种常见的方法,它使用在3'远端的逆转录酶将mRNA转录到核糖体6。然而,它不适用于进入核糖体的mRNA的5'结束。其他技术,包括单分子方法和荧光方法7,很难达到单nt分辨率。
我们开发了双DNA标尺测定,可以独特地确定核糖体-mRNA复合物中未发现的mRNA的入口和出口位置。 标尺DNA是DNA寡聚体,与核糖体发现的mRNA形成一定数量的碱基对(bp)的双面,无论mRNA的哪一端。然后,bp数精确地显示在易位期间mRNA上的核糖体位置。双工的bp数由它们从力引起的残余磁化光谱(FIRMS)8中获得的临界解离力决定。在 2-3 pN 力不确定的情况下,临界力足以提供单点分辨率。通过在DNA标尺上实现一个链接分子,可以探测核糖体对mRNA的消毒阻碍侧。因此,可以准确地解决不同的核糖体位移。我们已经成功地揭示了在易位9期间被抗生素捕获的mRNA的独特环状构象,并解决了在湿滑的mRNA序列10上共存的不同阅读帧。本文介绍了双标尺测定的细节,包括核糖体复合物的制备、玻璃玻片的表面功能化、核糖体复合物的固定性及其与磁性标记DNA标尺的杂交分子、磁检测和力谱分析。

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Lock-in Amplifier</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">SR530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Lock-in Amplifier</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">SR830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Laser</td>
			<td style="width:239px;">Newport</td>
			<td style="width:161px;">TLB-6918-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Function generator</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">DS345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Photo detectors</td>
			<td style="width:239px;">Thorlabs</td>
			<td style="width:161px;">DET36A</td>
			<td></td>
		</tr>
	</tbody>
</table>

dual dna rulers to study the mechanism of ribosome translocation with single-nucleotide resolution

核糖体易位是指在mRNA上核糖体运动,正好由三个核苷酸(nt),这是蛋白质合成的中心步骤。为了研究其机制,有两个基本的技术要求。首先是单nt分辨率,它可以解决帧移位的正常移位问题,在此期间,核糖体移动的分辨率超过 3 nt。第二种是探测mRNA的入口和出口两侧的能力,以便阐明易位的整体情况。我们报告双DNA标尺测定,该测定基于DNA-mRNA复式的关键解离力,通过力诱导的残余磁化光谱(FIRMS)获得。 使用 2-4 pN 力分辨率时,双标尺测定足以区分不同的易位步骤。通过在探测DNA上实现长链接器,它们可以到达核糖体另一侧的mRNA,从而确定双方的mRNA位置。因此,双标测定特别适合研究核糖体易位和核酸运动。我们显示了一个代表性的结果,表明环mRNA构象和解决帧移位的正常移位。

图1显示了主要部件的检测方案和照片。磁探测是通过原子磁力计使用扫描方案(图1A)13实现的。样品放置在安装在直线电机上的杆上。电机将样品输送到磁屏蔽内的原子传感器,然后返回原始站点进行卸载。原子磁力计在样品扫描过程中检测磁信号,并产生信号跟踪,当样品最接近传感器时,最大信号。图 1</stron...

Watch this Scientific Journal Video about Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution at JoVE.com

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

开发双DNA标尺测定,以确定核糖体易位过程中的mRNA位置,该位置依赖于形成DNA-mRNA复面的解离力。具有单核苷酸分辨率和达到mRNA两端的能力,可为核糖体易位提供机械见解,并探测其他核酸位移。

双DNA标尺研究单核苷酸分辨率的核糖体易位机制

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

双DNA标尺测定可以为核糖体易位提供机械见解，并探寻其他核酸位移。我们的双DNA标尺测定是目前唯一能够客观地探测核糖体复合物中mRNA入口和出口两侧的单核苷酸分辨率的方法。类似的方法也用于测量化疗药物的DNA结合强度和选择性。该方法的可视化演示是我们直观地展示我们技术优势并帮助其他研究人员进一步开发到更多应用中的好机会。首先，在中心钻一个尺寸为四毫米、三毫米、两毫米的立方体，用苯乙烯带制作塑料样品井。然后，使用环氧树脂，将一块长约五毫米的生物素涂层玻璃粘在底部表面。将20微升，每毫升0.25毫克水溶液加入样品中，在室温下孵育40分钟。然后用TAM10缓冲液冲洗样品两次。要固定在没有抗生素的核糖体复合物上，首先从样品井中去除缓冲液，并在样品井中加入20微升0.1微摩尔MF-Pre或MF-后核糖复合物。在37摄氏度下孵育一小时，然后用TAM10缓冲液冲洗一次。核糖体复合物现在通过mRNA上的五主要端生物素与表面的链球菌结合。在使用抗生素的实验中，用一微升4毫摩尔新霉素，37摄氏度，孵育9微升的MF-Pre复合物，10分钟。然后结合在管 0.6 微升 60 微摩尔 EFG， 0.8 微升 100 微摩尔 GTP、0.8 微升 100 毫摩尔 PEP、0.3 微升每毫升丙酮酸激酶 1.3 毫克、6.43 微升 TAM10 缓冲液和 1 微升 5 毫摩尔富西酸。在37摄氏度下孵育20分钟。然后将两种混合物混合，在37摄氏度下孵育5分钟。进行其他抗生素实验类似，浓度为0.2毫摩尔维奥霉素、0.4毫摩尔湿霉素B和0.25毫摩尔富西酸。对于帧移位研究，重复用于涉及滑滑图案 U6A 的 MFNF-Pre 复合物的孵化。包括富西酸和新霉素的抗生素。需要足够的时间来确保混合过程的完成。还建议使用TAM10缓冲液与实际一摩尔氯化钠来维持一个均匀系统。孵育后，从样品井中去除缓冲液。加入20微升的一微摩尔生物基化探测DNA链，并在室温下孵育过夜。早晨，用TAM10缓冲液冲洗一次成形的DNA/mRNA双工。从样品井中拆下缓冲液。然后将20微升，每毫升0.5毫克，在样品中加入0.5毫克的石丁涂层磁珠，并在室温下孵育两小时。之后，小心地将样品插入支架中，并将其放入离心机中，以 84 倍 g 的电流从表面清除自由磁性颗粒五分钟。打开激光。然后按下电源按钮。将灵敏度调整到 500 毫伏，等待约两个小时以预热和稳定原子磁力计。要设置原子磁力计，请运行仪器控制软件，将电机移动模式设置为"噪音"，将默认位置设置为零。按前面板上的锁。将灵敏度调整回 200 毫伏。调整激光的电流和电压，找到适当的谐振峰值和信噪比电平，以获得最佳信号分辨率。按前面板上的"扫描"。确保振幅宽度比高于 0.5，相位值小于宽度小于 170 赫兹的 5 度。将另一个 SR530 锁定放大器的输出插入激光器的反馈以锁定其频率。插入功能发生器 ATF20B，输入 500 毫伏峰值的方波峰值，100 毫赫作为将电流转换为磁性信号的参考信号。然后拔下功能发生器。将电机移动模式设置为双向，将默认位置设置为 260 毫米。检查原子传感器的温度控制器的状态，以确保温度在 37 摄氏度左右。直接运行程序，无需加载样品即可测量空样品支架的信号，检查整个系统的稳定性两次。要磁化样品，请轻轻使用钳子将样品从支架中取出，并在磁化阶段放置两分钟。然后将样品放回样品架中，以335.4倍g离心20分钟。去除非特异性磁性颗粒。接下来，用钳子将样品装到电机上。单击"锁定"前面板以运行程序。同时，使用钳子将另一个样品装入支架中，然后放入离心机。使涂层玻璃面朝向离心机的中心，以 335.4 倍 g 启动离心 4 分钟。当电机返回零时，大约五分钟后，单击"前面板上的保存"。小心地使用钳子将一个样品从电机转移到离心机支架上。通过将离心速度提高 100 rpm 或类似步长来施加更强的力。将样品交换到离心机。交替处理，每次逐渐增加力。在 10 到 12 个数据点后获得完整的力谱。完成所有计划的实验后，关闭设备，按相反的顺序运行，因为它已打开。从支架中取出样品，并将其浸入乙醇中，用于清洁和将来使用。在磁珠污染的情况下，用丙酮清洁样品架。在此协议中，核糖体复合物通过mRNA的五个底端固定在表面，使探测DNA标尺的五等和三等面都易于接近。当五个底端的连链接器超过50胸腺素时，检测到强磁信号，表明DNA/mRNA双面的成功形成。通过改变DNA上的核苷酸数量，分别实现了三等和五等面分离力与双面长度的相关性。在没有抗生素的情况下，从MF-Pre到MF-Post的正常移位结果表明，核糖体由三个核苷酸向两侧的三个主要端移动。MF-Pre分别携带了一个流浪的fMet-tRNA和苯丙氨酸-tRNA，而MF-Post在E和P站点分别携带了fMet-tRNA和苯丙氨酸-tRNA，并空出的一个 A站点。然而，当富西迪奇酸和二恶英抗生素都存在时，核糖体在五主要侧只移动一个核苷酸，而在三质一侧只移动两个核苷酸。洗掉抗生素后，正常易位发生，五等和三等两侧的三个核苷酸运动就证明。磁化样品时，涂层表面应接近并垂直离开磁铁。去除非特异性磁性粒子对数据的准确性和质量非常重要。我们的技术为研究核酸的分子位移提供了高分辨率和普遍适用的方法，这种位移在分子生物学中是广泛遇到的。

Research

JoVE Journal

Biochemistry

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

在质膜用新型荧光探针磷脂酰-4,5-二磷酸的单分子超分辨率成像

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

科研

生物化学

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

用限制性酶来测定染有菁染料的 DNA

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

单分子荧光能量转移研究核糖体蛋白质合成

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

双色荧光互相关光谱研究活细胞中蛋白质-蛋白质相互作用和蛋白质动力学

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

CD Spectroscopy to Study DNA-Protein Interactions

CD光谱学研究DNA-蛋白质相互作用

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...