双DNA标尺研究单核苷酸分辨率的核糖体易位机制
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Styrene Strip | City of Industry | MS-861 | |
| Glass slides | Evaporated Coatings | 60.0 × 4.0 × 0.3 mm3 | |
| Acetic acid | Millipore Sigma | A6283-500ML | |
| 3-Aminopropyltriethoxysilane | UCT specialties | 21400088 | |
| mPEG-SVA | Laysan Bio | 154-82 | |
| Biotin-PEG-SVA | Laysan Bio | 152-84 | |
| Sodium bicarbonate | Millipore Sigma | S5761-500G | |
| Epoxy glue | Devcon | 31345 | |
| Streptavidin | ThermoFisher | 434301 | |
| Fusidic Acid | Millipore Sigma | F0756-1G | |
| Neomycin Sulfate | Millipore Sigma | 1458009 | |
| Viomycin Sulfate | Millipore Sigma | 1715000 | |
| Hygromycin | invitrogen | 10687-010 | |
| Tris-HCl | Millipore Sigma | T5941-100G | |
| Magnesium acetate | Millipore Sigma | M5661-50G | |
| Ammonium chloride | Millipore Sigma | A9434-500G | |
| Potassium chloride | Millipore Sigma | P9333-500G | |
| EDTA | GIBCO | 774750 | |
| 2-mercaptoethanol | Millipore Sigma | M6250-500ML | |
| Tween20 | Millipore Sigma | P1379-250ML | |
| GTP | Millipore Sigma | G8877-100MG | |
| PEP | Millipore Sigma | P7127-100MG | |
| Pyruvate Kinase | Millipore Sigma | P1506-5KU | |
| Sucrose | Millipore Sigma | S7903-5KG | |
| Dynabeads M-280 Streptavidin | ThermoFisher | 11205D | |
| mRNA Oligo | Integrated DNA Technologies | 133899727 | 5′-Bio- CAA CUG UUA AUU AAA UUA AAU UAA AAA GGA AAU AAAA AUG UUU AAU UUU UUA GGG CGC AAU CUA CUG CUG AAC UC-3′ |
| DNA Oligo | Integrated DNA Technologies | 157468630 | 3ʹ- TAA TTT AAT TTA ATT TTT CGA AAU AT50/TEGBio/-5ʹ |
| DNA Oligo | Integrated DNA Technologies | 164845370 | 3ʹ-AAT TTA ATT TTT CCT TTA AAA AT50/TEGBio/-5’ |
| DNA Oligo | Integrated DNA Technologies | 157468628 | 3ʹ-AAA ATC CCG CGT TAG AAC UGG GG/TEGBio/-5’ |
| DNA Oligo | Integrated DNA Technologies | 163472705 | 3ʹ-CCG CGT TAG ATG ACG AGA ACG GG/TEGBio/-5’ |
| DNA Oligo | Integrated DNA Technologies | 138678130 | 3ʹ-AGA TGA CGA CTT CTC GGG/TEGBio/-5’ |
| DNA Oligo | Integrated DNA Technologies | 138678131 | 3ʹ-T AGA TGA CGA CTT CTC GGG/TEGBio/-5’ |
| DNA Oligo | Integrated DNA Technologies | 138678132 | 3ʹ-TT AGA TGA CGA CTT CTC GGG/TEGBio/-5ʹ |
| DNA Oligo | Integrated DNA Technologies | 138678133 | 3ʹ-GTT AGA TGA CGA CTT CTC GGG/TEGBio/-5’ |
| Centrifuge | Eppendorf | 5427R | |
| Micro Ultracentrifuge | Hitachi | CS150FNX | |
| Vortex mixer | VWR | VM-3000 | |
| Lock-in Amplifier | Stanford Research Systems | SR530 | |
| Lock-in Amplifier | Stanford Research Systems | SR830 | |
| Laser | Newport | TLB-6918-D | |
| Function generator | Stanford Research Systems | DS345 | |
| Photo detectors | Thorlabs | DET36A |
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