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基于荧光-mRNA 的实时分析方法的优化用于核糖体移位的精确动力学测量。

Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation.

机构信息

Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

出版信息

RNA Biol. 2021 Dec;18(12):2363-2375. doi: 10.1080/15476286.2021.1913312. Epub 2021 May 3.

DOI:10.1080/15476286.2021.1913312
PMID:33938388
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8632105/
Abstract

Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNA, with the natural dipeptidyl fMet-Phe-tRNA in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.

摘要

核糖体易位的动力学特征对于理解蛋白质合成中延伸的机制非常重要。在这里，我们通过在停流中用功能性三肽形成测定法对其进行校准，优化了流行的基于荧光 mRNA 的易位测定法。我们发现，从+1 位（mRNA+10）长 10 个碱基的荧光标记 mRNA 最适合两种测定法，因为它以与较长的 mRNA 相当的快速速率形成三肽，但在 mRNA 移动时会产生较大的荧光变化。接下来，我们在停流测定法中比较了常用的肽酰 tRNA 类似物 N-乙酰-Phe-tRNA 与天然二肽基 fMet-Phe-tRNA。该类似物的易位速度比天然二肽基 tRNA 慢约两倍，并且产生双相动力学。在较低温度和较高 Mg 浓度下，速率进一步降低，但随着伸长因子 G（EF-G）浓度的升高而改善，这显著提高了快速相的速率和幅度。总之，我们在这里提出了一种改进的实时测定法，用于监测天然和二肽基 tRNA 的 N-Ac 类似物的 mRNA 易位。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/fe5d302447c4/KRNB_A_1913312_F0005_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/666dabd0a7db/KRNB_A_1913312_UF0001_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/2c1495f1fd0b/KRNB_A_1913312_F0001_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/a57872d3a11a/KRNB_A_1913312_F0002_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/0e55e5609928/KRNB_A_1913312_F0003_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/40e1dc40ff21/KRNB_A_1913312_F0004_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/fe5d302447c4/KRNB_A_1913312_F0005_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/666dabd0a7db/KRNB_A_1913312_UF0001_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/2c1495f1fd0b/KRNB_A_1913312_F0001_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/a57872d3a11a/KRNB_A_1913312_F0002_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/0e55e5609928/KRNB_A_1913312_F0003_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/40e1dc40ff21/KRNB_A_1913312_F0004_OC.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/693d/8632105/fe5d302447c4/KRNB_A_1913312_F0005_OC.jpg

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本文引用的文献

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Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association.核糖体柄蛋白 L7/12 与 IF2 之间的补充电荷相互作用是快速亚基结合的关键。
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tRNA Fluctuations Observed on Stalled Ribosomes Are Suppressed during Ongoing Protein Synthesis.
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